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THE  LABORATORY  OF  PHYSIOLOGICAL 
CHEMISTRY 

SHEFFIELD  SCIENTIFIC  SCHOOL 

YALE  UNIVERSITY 


Collected  papers 
191 1-1912 


NEW  HAVEN,  CONNECTICUT 
U.  S.  A. 


THE  LABORATORY  OF  PHYSIOLOGICAL  CHEMISTRY 


RUSSELL  H.  CHITTENDEN 

Professor  of  Physiological  Chemistry 

LAFAYETTE  B.  MENDEL 

Professor  of  Physiological  Chemistry 

FRANK  P.  UNDERHILL 

Assistant  Professor  of  Physiological  Chemistry 


CONTENTS 

The  Influence  of  Urethane  in  the  Production  of  Glycosuria  in  Rabbits 
after  the  Intravenous  Injection  of  Adrenalin.  By  Frank  P. 
Undrrhill.     (From  the  Journal  of  Biological  Chemistry,  1911, 

ix,  13.) 

Mucic  Acid  and  Intermediary  Carbohydrate  Metabolism.  By  William 
C.  Rose.    (From  the  Journal  of  Biological  Chemistry,  1911,  x,  123.) 

Experimental  Studies  on  Creatine  and  Creatinine. — I.  The  Role  of  the 
Carbohydrates  in  Creatine — Creatinine  Metabolism.  By  Lafayette 
B.  Mendel  and  William  C.  Rose.  (From  the  Journal  of  Bio- 
logical Chemistry,  191 1,  x,  213.) 

Experimental  Studies  on  Creatine  and  Creatinine. — II.  Inanition  and  the 
Creatine  Content  of  Muscle.  By  Lafayette  B.  Mendel  and  Wil- 
liam C.  Rose.    (From  the  Journal  of  Biological  Chemistry,  1911, 

x,  255.) 

Experimental  Studies  on  Creatine  and  Creatinine. — III.  Excretion  of 
Creatine  in  Infancy  and  Childhood.  By  William  C.  Rose.  (From 
the  Journal  of  Biological  Chemistry,  1911,  x,  265.) 

Studies  in  Nutrition. — I.  The  Utilization  of  the  Proteins  of  Wheat. 
By  Lafayette  B.  Mendel  and  Morris  S.  Fine.  (From  the  Journal 
of  Biological  Chemistry,  1911,  x,  303.) 

Studies  in  Nutrition. — II.  The  Utilization  of  the  Proteins  of  Barley. 
By  Lafayette  B.  Mendel  and  Morris  S.  Fine.  (From  the  Journal 
of  Biological  Chemistry,  191 1,  x,  339.) 

Studies  in  Nutrition. — III.  The  Utilization  of  the  Proteins  of  Corn. 
By  Lafayette  B.  Mendel  and  Morris  S.  Fine.  (  From  the  Journal 
of  Biological  Chemistry,  191 1.  x,  345.) 

Studies  in  Nutrition. — IV.  The  Utilization  of  the  Proteins  of  the 
Legumes.  By  Lafayette  B.  Mendel  and  Morris  S.  Fine.  (From 
the  Journal  of  Biological  Chemistry,  1912.  x,  433.) 

Studies  in  Nutrition. — V.  The  Utilization  of  the  Proteins  of  Cotton  Seed. 
By  Lafayette  B.  Mendel  and  Morris  S.  Fine.  (  From  the  Journal 
of  Biological  Chemistry,  1912,  xi.  1.) 

Studies  in  Nutrition. — VI.  The  Utilization  of  the  Proteins  of  Extrac- 
tive-Free Meat  Powder;  and  the  Origin  of  Fecal  Nitrogen.  By 
Lafayette  B.  Mendel  and  Morris  S.  Fine.  (From  the  Journal  of 
Biological  Chemistry,  1912,  xi,  5.) 


r?  4  ^ 


I 


CONTENTS 


III 


Studies  in  Carbohydrate  Metabolism. — I.  The  Influence  of  Hydrazine 
upon  the  Organism,  with  Special  Reference  to  the  Blood  Sugar 
Content.  By  Frank  P.  Underbill.  (From  the  Journal  of  Bio- 
logical Chemistry,  ion,  x,  150.) 

Studies  in  Carbohydrate  Metabolism. —  tl.  The  Prevention  and  Inhibi- 
tion of  Pancreatic  Diabetes.  By  Frank  P.  Underbill  and  Morris 
S.  Fine.    (From  the  Journal  of  Biological  Chemistry,  1911,  x,  271.) 

The  Production  of  Glycosuria  as  a  Result  of  the  Intravenous  Injection 
of  Witte's  Peptone.  By  Yandell  Henderson  and  Frank  P. 
Underhill.  (From  the  Proceedings  of  the  Society  for  Experi- 
mental Biology  and  Medicine,  191 1,  viii,  80.) 

The  Behavior  of  Fat-Soluble  Dyes  in  the  Organism.  By  Lafayette  B. 
Mendel  and  Amy  L.  Daniels.  (From  the  Proceedings  of  the 
Society  for  Experimental  Biology  and  Medicine,  191 1,  viii,  126.) 

The  Production  of  Glycosuria  by  Adrenalin  in  Thyroidectomized  Dogs. 
By  Frank  P.  Underhill.  (From  the  American  Journal  of 
Physiology,  1911,  xxvii,  331.) 

The  Metabolism  of  Dogs  with  Functionally  Resected  Small  Intestine. 
By  Frank  P.  Underhill  (with  the  collaboration  of  Chester  J. 
Stedman  and  Jessamine  Chapman).  (From  the  American  Journal 
of  Physiology,  1911,  xxvii,  366.) 

Acapnia  and  Glycosuria.  By  Yandell  Henderson  and  Frank  P.  Under- 
hill.    (From  the  American  Journal  of  Physiology,  191 1,  xxviii, 


Nutrition  Investigations  on  the  Carbohydrates  of  Lichens,  Algae,  and 
Related  Substances.  By  Mary  Davies  Swartz.  (From  the  Trans- 
actions of  the  Connecticut  Academy  of  Arts  and  Sciences,  191 1, 
xvi,  247.) 

A  Consideration  of  Some  Chemical  Transformations  of  Proteins  and 
their  Possible  Bearing  on  Problems  in  Pathology.  By  Frank  P. 
Underhill.     (From  the  Archives  of  Internal  Medicine A  191 1,  viii, 


The  Role  of  Different  Proteins  in  Nutrition  and  Growth.  By  Thomas 
B.  Osborne  and  Lafayette  B.  Mendel.  (From  Science,  191 1, 
xxxiv,  722.) 

The  Action  of  Salts  of  Choline  on  Arterial  Blood  Pressure.  By 
Lafayette  B.  Mendel,  Frank  P.  Underhill,  and  R.  R.  Renshaw. 
(From  the  Journal  of  Pharmacology  and  Experimental  Therapeutics, 
1912,  iii,  649.) 

The  Influence  of  Tartrates  upon  Phlorhizin  Diabetes.  By  Frank  P. 
Underhill.  (From  the  Proceedings  of  the  Society  for  Experi- 
mental Biology  and  Medicine,  1912,  ix,  123.) 

The  Haemagglutinating  and  Precipitating  Properties  of  the  Bean 
(Phaseolus) .  By  Edward  C.  Schneider.  (From  the  Journal  of 
Biological  Chemistry,  1912,  xi,  47.) 

The  Value  of  Inulin  as  a  Foodstuff.  By  Howard  B.  Lewis.  (From  the 
Journal  of  the  American  Medical  Association,  1912,  lviii,  1176.) 

The  Influence  of  Cocaine  upon  Metabolism  with  Special  Reference  to 
the  Elimination  of  Lactic  Acid.  By  Frank  P.  Underhill  and 
Clarence  L.  Black.  (From  the  Journal  of  Biological  Chemistry, 
1912,  xi,  235.) 


275.) 


356.) 


ADVERSITY  Of 


iv 


CONTENTS 


The  Physiological  Action  of  Some  Pyrimidine  Compounds  of  the  Barbi- 
turic Acid  Series.  By  Israel  S.  Kleiner.  (From  the  Journal  of 
Biological  Chemistry,  1912,  xi,  443.) 

The  Influence  of  Sodium  Tartrate  upon  the  Elimination  of  Certain 
Urinary  Constituents  during  Phlorhizin  Diabetes.  By  Frank  P. 
Underhill.  (From  the  Journal  of  Biological  Chemistry,  1912,  xii, 
115.) 

Feeding  Experiments  with  Fat-Free  Food  Mixtures.  By  Thomas  B. 
Osborne  and  Lafayette  B.  Mendel  (with  the  cooperation  of 
Edna  L.  Ferry).    (From  the  Journal  of  Biological  Chemistry,  1912, 

xii,  81.) 

The  Role  of  Gliadin  in  Nutrition.  By  Thomas  B.  Osborne  and  Lafay- 
ette B.  Mendel  (with  the  cooperation  of  Edna  L.  Ferry).  (From 
the  Journal  of  Biological  Chemistry,  1912,  xii,  473.) 

Maintenance  Experiments  with  Isolated  Proteins.  By  Thomas  B. 
Osborne  and  Lafayette  B.  Mendel  (with  the  cooperation  of 
Edna  L.  Ferry).  (From  the  Journal  of  Biological  Chemistry, 
1912,  xiii,  233.) 

A  Study  of  the  Mechanism  of  Phlorhizin  Diabetes.  By  Frank  P. 
Underhill.     (From  the  Journal  of  Biological  Chemistry,  1912, 

xiii,  15.) 

The  Behavior  of  Fat-Soluble  Dyes  and  Stained  Fat  in  the  Animal  Organ- 
ism. By  Lafayette  B.  Mendel  and  Amy  L.  Daniels.  (From  the 
Journal  of  Biological  Chemistry,  1912,  xiii,  71.) 

Beobachtungen  iiber  Wachstum  bei  Fiitterungsversuchen  mit  isolierten 
Nahrungssubstanzen.  By  Thomas  B.  Osdorne  and  Lafayette  B. 
Mendel  (with  the  cooperation  of  Edna  L.  Ferry).  (From  the 
Zeitschrift  fur  physiologische  Chemie,  1912,  lxxx,  307.) 

The  Behavior  of  Some  Hydantoin  Derivatives  in  Metabolism. — I.  Hydan- 
toin  and  Ethyl  Hydantoate.  By  Howard  B.  Lewis.  (From  the 
Journal  of  Biological  Chemistry,  1912,  xiii,  347.) 

The  following  additional  papers  from  the  Laboratory  are  not  included 
in  this  volume : 

Theorien  des  Eiweissstoffwechsels  nebst  einigen  praktischen  Konsequenzen 
derselben.  By  Lafayette  B.  Mendel.  Ergebuissc  der  Physiologie, 
1911,  xi,  418. 

Feeding  Experiments  with  Isolated  Food-Substances.  By  Thomas  B. 
Osborne  and  Lafayette  B.  Mendel  (with  the  cooperation  of 
Edna  L.  Ferry).  Carnegie  Institution  of  Wasliington,  Publication 
156,  Parts  I  and  II.    1912.    pp.  138. 

Zur  Wirkung  intravenoser  Einspritzungen  von  Konzentrierten  Salz-und 
Zuckerlosungen.  By  Frank  P.  Underhill.  Archiv  fur  experi- 
mentellc  Pathologie  unci  Pharmakologie ,  191 1,  lxix,  407. 

Ein  StofTwechselkafig  und  Fiitterungsvorrichtungen  fiir  Ratten.  By 
Thomas  B.  Osborne  and  Lafayette  B.  Mendel.  Zeitschrift 
fiir  biologische  Technik  und  Methodik,  1912,  ii,  313. 

Biochemical  and  Bacteriological  Studies  on  the  Banana.  By  E.  Monroe 
Batley.  Journal  of  the  American  Chemical  Society,  1912,  xxxiv, 
1706. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  IX,  No.  1,  1911. 


THE  INFLUENCE  OF  URETHANE  IN  THE  PRODUCTION 
OF  GLYCOSURIA  IN  RABBITS  AFTER  THE  IN- 
TRAVENOUS INJECTION  OF  ADRENALIN. 

By  FRANK  P.  UNDERHILL. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  December  8,  1910.) 

The  vast  accumulation  of  literature  relative  to  adrenalin  gly- 
cosuria contains  very  few  records  of  attempts  to  demonstrate  a 
quantitative  relationship  between  adrenalin  administration  and 
sugar  elimination.  For  the  most  part  investigators  have  been 
content  with  the  knowledge  that  a  certain  quantity  of  adrenalin 
injected  subcutaneously  or  intraperitoneally  almost  invariably 
causes  the  appearance  in  the  urine  of  significant  quantities  of 
dextrose.  Moreover,  it  has  been  generally  accepted  that  adrenalin 
given  by  mouth  entirely  fails  to  provoke  glycosuria  and  that  the 
intraperitoneal  administration  gives  rise  to  a  greater  sugar  excre- 
tion than  the  introduction  of  the  drug  directly  into  the  circulation. 
What  relationship  exists  between  the  quantity  of  adrenalin  injected 
and  the  sugar  eliminated,  and  how  this  relation  may  vary  with 
change  in  the  manner  of  adrenalin  introduction  are  questions  of 
some  importance  since  adrenalin  effects  are  constantly  employed 
in  the  study  of  problems  having  a  far  reaching  significance. 

Perhaps  the  most  suggestive  investigation  in  this  particular 
direction  is  the  recent  communication  of  Ritzmann1.  He  has  shown 
that  the  degree  of  glycosuria  is  dependent  upon  the  quantity  of 
adrenalin  present  in  the  blood  at  any  given  moment.  So  long  as 
adrenalin  is  present  in  the  blood  sugar  in  the  urine  is  in  order  but 

1  Ritzmann:  Arch.  f.  exp.  Path.  u.  Pharmakol.,  lxi,  p.  231,  1909.  See  also 
Straub:  Munch,  med.  Wochenschr.,  1909,  No.  10;  Pollak:  Arch.  f.  exp. 
Path.  u.  Pharmakol,  lxi,  p.  376,  1909. 

13 


14 


Urethane  and  Adrenalin  Glycosuria 


glycosuria  ceases  as  soon  as  adrenalin  disappears  from  the  circula- 
tion and  almost  immediately  reappears  when  the  drug  is  again 
introduced.  Extremely  dilute  adrenalin  solutions  are  potent  in 
eliciting  a  relatively  large  excretion  of  sugar.  There  exists,  accord- 
ing to  Ritzmann,  a  direct  relationship  between  the  quantity  of 
adrenalin  introduced  into  the  circulation  and  the  amount  of  sugar 
eliminated  in  the  urine,  and  for  each  rate  of  adrenalin  injection 
there  is  a  corresponding  grade  of  glycosuria.  Furthermore,  adrena- 
lin administered  subcutaneously  is  not  capable  of  inducing  as 
much  sugar  to  appear  in  the  urine  as  a  much  smaller  quantity  of  the 
drug  introduced  intravenously.  In  Ritzmann's  experiments  cats 
and  rabbits  were  employed  and  the  adrenalin  was  introduced  into 
the  jugular  or  femoral  veins  by  a  modification  of  the  Kretchmer 
method.  The  injections  were  made  with  the  animals  under  anaes- 
thesia. In  general  when  rabbits  were  used  narcosis  was  produced 
-with  urethane  given  by  stomach  sound.  Urine  was  collected 
through  a  permanent  catheter. 

In  the  course  of  an  investigation  it  became  desirable  to  make  use 
of  Ritzmann's  observations  and  trials  were  made  in  order  to  deter- 
mine whether  in  our  hands  entirely  corroboratory  results  could 
be  obtained.  Our  method  of  introducing  the  drug  directly  into 
the  circulation  of  the  experimental  animal  (the  rabbit)  was  that 
indicated  in  a  former  paper,1  that  is,  adrenalin  (Parke,  Davis  and 
Company),  suitably  diluted  with  0.9  per  cent  sodium  chloride 
solution,  was  injected  into  the  ear  vein  under  pressure.  This  obvi- 
ated the  necessity  of  narcosis.  Urine  was  obtained  at  the  desired 
intervals  by  compression  of  the  bladder  through  the  body  wail. 
Our  experimental  conditions  conformed  in  every  other  respect  with 
those  of  Ritzmann. 

In  spite  of  the  harmony  existing  between  the  experimental  con- 
ditions of  Ritzmann's  investigation  and  our  own  all  attempts 
to  provoke  glycosuria  by  intravenous  injections  of  dilute  solutions 
of  adrenalin  into  normal  rabbits  failed.  Two  experiments  are 
given  below  in  detail  which  will  serve  as  typical  examples  of  a 
large  number  of  similar  observations.  Table  1  shows  the  results 
obtained  in  an  attempt  to  duplicate  Experiment  4  (p.  239)  of 
Ritzmann  while  the  data  in  Table  2  are  those  yielded  in  an  en- 

1Underhill  and  Closson:  Amer.  Journ.  of  Physiol.,  xv,  p.  321,  1906. 


Frank  P.  Underhill 


deavor  to  duplicate  Ritzmann's  Experiment  15  (p.  240).  In 
Ritzmann's  Experiment  4,  0.18  gram  dextrose  was  present  in  the 
urine  after  the  introduction  of  75  cc.  of  1 :500000  adrenalin  solu- 
tion in  40  minutes,  whereas  in  the  present  investigation,  Table 
1,  no  sugar  appeared  after  nearly  200  cc.  had  been  injected  at  the 
same  rate.  It  is  shown  in  Experiment  15  of  Ritzmann's  work  that 


TABLE  1. 

Female  rabbit  of  2JfiO  grams  weight.  The  urine  in  the  bladder  contained 
no  reducing  substances.    Adrenalin  solution,  1:500000. 


TIME. 

ADRENALIN  INJECTED. 

URINE  EXCRETED. 

REDUCTION  TEST.* 

CC. 

CC. 

11.22 

0 

0 

11.42 

41 

25 

negative 

12.02 

35 

10 

negative 

12.22 

40 

8 

negative 

12.42 

32 

10 

negative 

1.02 

50 

20 

negative 

*With  Benedict's  reagent.   Benedict:  Journal  of  Biological  Chemistry;  1908,  v.  p.  485. 


TABLE  2. 

Male  rabbit  of  2200  grams  weight.    Urine  in  bladder  did  not  reduce.  Adrenalin 
solution  employed,  1:250000. 


TIME. 

ADRENALIN  INJECTED. 

URINE  EXCRETED. 

REDUCTION  TEST. 

CC. 

CC. 

3.19 

0 

3.39 

49  • 

0 

negative 

4.05 

34 

5 

negative 

4.27 

35 

20 

negative 

4.47 

50 

17 

negative 

4.58 

15 

7 

negative 

5.18 

50 

10 

negative 

5.39 

50 

18 

negative 

6.00 

50 

11 

negative 

0.28  gram  sugar  was  present  in  the  urine  when  49  cc.  of  1:250000 
adrenalin  solution  had  been  injected  in 20 minutes.  Our  introduc- 
tion of  333  cc.  (Table  2)  of  the  same  strength  and  at  the  same 
rate  of  injection  did  not  induce  any  glycosuria. 

In  an  endeavor  to  account  for  the  discrepancy  between  Ritz- 
mann's results  and  our  own  control  experiments  were  carried  through 


1 6  Urethane  and  Adrenalin  Glycosuria 


without  suggesting  a  probable  explanation.  Finally  observations 
were  made  upon  animals  that  had  received  urethane  by  mouth. 
All  experiments  in  which  sufficient  urethane  had  been  given  yielded 
results  in  entire  accord  with  those  of  Ritzmann.  Positive  results  were 
obtained  invariably  only  when  sufficient  urethane  had  been  intro- 
duced. The  quantity  of  urethane  necessary  appears  to  be  about 
one  gram  per  kilo  of  body  weight.  Smaller  quantities  will,  however, 
frequently  furnish  positive  results  but  the  smaller  quantities  cannot 
be  relied  upon,  whereas  with  doses  of  one  gram  per  kilo  not  a  single 
experiment  failed  to  induce  glycosuria. 

Table  3  presents  data  obtained  upon  an  animal  without  ure- 
thane narcosis  and  in  Table  4  may  be  found  results  from  the  same 
animal  under  urethane  narcosis.    Urethane  alone  is  incapable  of 

TABLE  3. 

Male  rabbit  of  2000  grains  weight.  Bladder  empty  when  tested.  1.5  grams  ure- 
thane in  20  cc.  water  by  stomach  tube.  Adrenalin  solution  employed, 
1:250000. 


TIME. 

ADRENALIN  INJECTED. 

URINE  EXCRETED 

REDUCTION  TEST. 

CC. 

CC. 

3.25 

0 

3.45 

38 

0 

negative 

4.05 

33 

0 

negative 

4.25 

43 

2 

negative 

4.45 

42 

3 

negative 

5.05 

45 

6 

negative 

5.25 

45 

26 

negative 

5.45 

45 

23 

negative 

inducing  glycosuria  and  negative  results  are  also  yielded  when  so- 
dium chloride  is  introduced  into  a  urethane  narcotized  rabbit  in  the 
quantities  and  at  the  rate  employed  in  the  adrenalin  experiments. 
From  these  observations  it  would  therefore  appear  that  urethane 
renders  the  rabbit  organism  unusually  sensitive  to  the  glycosuria- 
inducing  action  of  adrenalin.  It  has  been  demonstrated  by  Froh- 
lich  and  LoewiHhat  cocaine  causes  the  organism  of  the  cat  and  dog 
to  become  more  sensitive  to  adrenalin  with  respect  to  its  influence 
upon  blood  pressure,  salivary  secretion  and  mydriasis.  It  is  possible 


^rohlich  and  Loewi :  Arc h.  f.  exp.  Path.  u.  Pharmakol.,  lxii,  p.  159,  1910. 


Frank  P.  Underhill 


17 


that  adrenalin  plays  a  role  in  adrenalin  glycosuria  somewhat  analo- 
gous to  that  of  cocaine  observed  by  the  above  mentioned  authors. 
Moreover  it  is  possible  that  other  narcotics  and  anaesthetics  may 
exercise  an  influence  similar  to  that  of  urethane  in  this  and  other 
forms  of  experimental  glycosuria. 

In  another  portion  of  Ritzmann's  paper  there  appears  a  compari- 
son of  the  influence  of  adrenalin  when  administered  subcutaneously 
and  intravenously.  It  is  shown  that  a  very  small  quantity  of 
adrenalin  injected  intravenously  will  cause  very  much  more  sugar 
to  appear  in  the  urine  than  a  much  larger  quantity  of  adrenalin 
introduced  subcutaneously.  This  comparison  seems  hardly  fair 
in  view  of  the  influence  of  urethane  noted  above  since  in  Ritz- 
mann 's  experiments  the  narcotic  was  employed  only  when  adrena- 

TABLE  4. 


Same  animal  that  was  employed  in  previous  experiment — four  days  later. 
Urine  in  bladder  gave  no  reduction,  2.0  grams  urethane  in  25  cc.  water  by 
mouth.   Adrenalin  employed,  1  -.250000. 


TIME. 

ADRENALIN  INJECTED 

URINE  EXCRETED. 

REDUCTION  TEST. 

CC. 

CC. 

10.46 

0 

11.05 

34 

10 

negative 

11.25 

39 

5 

negative 

11.45 

38 

4 

negative 

12.05 

38 

22 

Strong 

12.25 

34 

20 

very  strong 

lin  was  given  intravenously.  From  a  consideration  of  the  above 
observations  concerning  the  role  played  by  urethane  in  the  pro- 
duction of  glycosuria  after  intravenous  injections  of  dilute  solu- 
tions of  adrenalin  it  appeared  desirable  to  determine  whether  in 
the  non-narcotized  animal  a  definite  quantity  of  adrenalin  injected 
intravenously  in  dilute  solutions  would  yield  more  sugar  in  the 
urine  than  the  same  quantity  of  adrenalin  administered  subcutane- 
ously in  the  dilution,  1 :1000.  Table  5  shows  the  results  of  four  such 
experiments.  The  rabbits  were  maintained  under  constant  con- 
ditions of  diet  throughout  so  that  the  divergences  in  sugar  elimina- 
tion can  not  be  ascribed  to  such  an  origin.  Neither  can  they  be 
attributed  to  lack  of  glycogen  in  the  body  since  a  lapse  of  time  was 
allowed  between  the  injections  sufficient  for  the  production  of  a 


1 8  Urethane  and  Adrenalin  Glycosuria 


new  store  of  glycogen.  From  an  inspection  of  Table  5  it  will  be  at 
once  apparent  that  the  intravenous  administration  of  adrenalin 
in  the  dilution  employed  is  far  less  potent  in  the  non-narcotized 
rabbit  than  the  usual  subcutaneous  injection  with  respect  to  the 
appearance  of  sugar  in  the  urine.  These  results  are  in  direct  opposi- 
tion to  those  of  Ritzmann.  The  use  of  urethane  in  Ritzmann's 
experiments  is  undoubtedly  the  factor  responsible  for  our  failure 
to  corroborate  his  conclusions.  Our  experiments  also  indicate 
the  variation  in  the  quantity  of  sugar  eliminated  by  the  normal 
rabbit  maintained  under  constant  conditions  when  equal  doses  of 
adrenalin  are  subcutaneously  administered  at  different  periods, 
an  observation  which  has  been  corroborated  for  the  dog. 


TABLE  5. 


RABBIT. 

SUGAR  IN  URINE  AFTER  SUBCUTANEOUS 
INJECTION  OF  ONE  MILLIGRAM  ADRE- 
NALIN PER  KILO  IN  DILUTION  1:1000 

SUGAR  IN  URINE  AFTER  INTRAVENOUS 
INJECTION  OF  ONE  MILLIGRAM 
ADRENALIN  PER  KILO  IN 

First  injection 

Second  injection 

dilution  1:125000. 

1 

3.58 

4.17 

0.0 

2 

1.91 

3.50 

0.0 

3 

2.78 

3.39 

0.38 

4 

3.47 

4.93 

0.0 

CONCLUSIONS. 

Data  are  furnished  from  which  it  is  concluded  that  adrenalin 
introduced  in  very  dilute  solutions  (1:500000  to  1:125000)  fails  to 
induce  glycosuria  in  the  normal  rabbit.  On  the  other  hand,  when 
the  animal  is  under  the  influence  of  urethane  narcosis  these  dilute 
adrenalin  solutions  are  a  sufficient  stimulus  for  the  production  of 
glycosuria. 

From  these  observations.it  is  apparent  that  urethane  renders  the 
rabbit  organism  unusually  sensitive  to  the  glycosuria-inducing 
action  of  adrenalin.  The  subcutaneous  administration  of  adrena- 
lin in  the  usual  dilution  (1:1000)  to  normal  rabbits  is  far  more 
efficacious  in  causing  glycosuria  than  the  same  quantity  of  adrena- 
lin introduced  intravenously  in  much  greater  dilution. 

The  same  quantity  of  adrenalin  injected  subcutaneously  at  differ- 
ent periods  into  the  same  animal  under  constant  conditions  causes 
the  appearance  in  the  urine  of  variable  quantities  of  sugar. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  X,  No.  2,  1911 


MUCIC  ACID  AND    INTERMEDIARY  CARBOHYDRATE 
METABOLISM.1 

By  WILLIAM  C.  ROSE. 

(From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Conn.) 

(Received  for  publication,  July  16,  1911.) 


INTRODUCTORY. 


Various  views  have  been  debated  regarding  the  intermediate 
processes  through  which  the  carbohydrates  pass  in  metabolism  until 
they  are  finally  eliminated  in  the  form  of  their  end  products.  As 
might  be  expected  those  theories  fashioned  from  the  experience 
gained  in  the  study  of  the  fermentation  of  sugars  have  received  spe- 
cial attention.2  Thus  the  formation  of  lactic  acid  from  carbohy- 
drates through  the  agency  of  microorganisms  finds  a  counterpart 
in  the  occurrence  of  the  same  substance  in  the  tissues  and  secre- 
tions of  the  animal  body.  Metabolism  of  the  carbohydrate 
molecule  by  the  mechanism  here  suggested,  involves  an  early 
splitting  of  the  carbon  chain. 

A  distinctly  different  theory  demands  a  direct  oxidation  of  the 
carbohydrates  without  preliminary  cleavage  of  the  chain,  with 
the  formation  of  such  intermediate  products  as  glycuronic,  sac- 
charic and  oxalic  acids,  as  represented  in  the  following  formulas: 


CH2OH 
I 

(CHOH). 


COOH 
I 

(CHOH)4  — 


COOH 


(CHOH)4  — 


COOH 

I 


CHO 
Dextrose 


CHO 
Glycuronic 
Acid 


COOH 
Saccharic 
Acid 


COOH 
Oxalic 
Acid 


1 A  preliminary  report  of  this  investigation  was  presented  at  the  meet- 
ing of  the  American  Society  of  Biological  Chemists,  December,  1910; 
see  Proceedings,  this  Journal,  ix,  p.  xii,  1911. 

2  The  more  recent  aspects  of  this  subject  are  reviewed  by  Harden: 
Alcoholic  Fermentation,  1911. 

123 


124 


Metabolism  of  Mucic  Acid 


This  view  was  early  supported  by  Schmiedeberg  and  Meyer,1 
who  considered  glycuronic  acid  a  metabolic  oxidative  product  of 
dextrose,  on  account  of  the  related  chemical  structures  of  the  two 
substances,  and  the  ease  with  which  dextrose  is  converted  into 
glycuronic  acid  by  oxidizing  agents  outside  of  the  body.  This 
view  was  also  accepted  by  Emil  Fischer  and  Piloty.2  From  feed- 
ing experiments  with  thymotinpiperidid,  Hildebrandt3  concluded 
that  glycuronic  acid  may  be  formed  from  dextrose  in  the  animal 
organism.  He  claims  that  fatal  doses  of  this  base,  which  conju- 
gates with  glycuronic  acid,  exert  no  toxic  action  in  rabbits  if  the 
animals  have  previously  received  dextrose.  According  to  P. 
Mayer,4  the  elimination  of  glycuronic  acid  may  be  markedly  in- 
creased in  severe  disturbances  of  respiration,  such  as  dyspnoea, 
and  in  diabetes  mellitus.  The  observations  of  Mayer  are,  how- 
ever, not  substantiated  by  the  more  recent  investigations  of  Fen- 
nyvessy,5  and  the  statements  on  these  questions  are  contradictory. 
That  glycuronic  acid  is  not  a  product  of  sugar  metabolism  seems 
to  be  indicated  by  the  work  of  Loewi.6  This  investigator  fed 
dogs  with  camphor  during  phlorhizin  diabetes,  and  found  that 
although  enormous  quantities  of  campho-glycuronic  acid  were 
excreted,  the  sugar  elimination  was  only  very  slightly  diminished. 
A  much  larger  reduction  in  urinary  dextrose  ought  to  have  occurred 
if  it  had  been  the  mother-substance  of  glycuronic  acid.  Somewhat 
conflicting  views  in  regard  to  this  matter  are  given  by  Mandel  and 
Jackson7  and  by  Jackson.8 

In  experiments  upon  rabbits,  Mayer9  noted  an  increase  in  the 
output  of  oxalic  acid  after  the  introduction  of  glycuronic  acid, 
either  per  os  or  subcutaneously.  The  livers  of  animals  receiving 
glycuronic  acid  were  found  to  be  richer  in  oxalic  acid  than  those 

Schmiedeberg  and  Meyer,  H.:  Zeitschr.  f.  physiol.  XJhem.,  iii,  pp. 
422-50,  1879. 

2 Fischer  and  Piloty:  Ber.  d.  deutsch.  chem.  Gesellsch.,xxiv,  pp.  521-28, 
1891. 

3 Hildebrandt:  Arch.  f.  exp.  Path.  u.  Pharm.,  xliv,  pp.  278-316,  1900. 
4Mayer:  Zeitschr.  f.  klin.  Med.,  xlvii,  pp.  68-108,  1902. 
5Fennyvessy:  Arch,  internat.  d.  pharmacodyn.,  xii,  pp.  407-20,  1904. 
8Loewi:  Arch.  f.  exp.  Path.  u.  Pharm.,  xlvii,  pp.  56-67,  1902. 
7Mandel  and  Jackson:  Amer.  Journ.  of  Physiol.,  viii,  p.  xiii,  1902-03. 
8  Jackson:  Amer.  Journ.  of  Physiol.,  viii,  p.  xxxii,  1902-03. 
9 Mayer:  loc.  cit. 


William  C.  Rose 


125 


of  control  animals  which  had  been  similarly  fed  without  glyc  .ironic 
acid.  Autolytic  experiments  indicated  that  liver  extracts  con- 
tained enzymes  capable  of  producing  the  change  from  glycuronic 
to  oxalic  acid. 

Thierfelder1  has  shown  that  saccharic  acid  results  from  the  oxi- 
dation of  glycuronic  acid  in  vitro,  but  there  is  no  direct  evidence 
that  this  change  can  occur  in  the  animal  organism.  Mayer2 
reported  an  increase  in  urinary  oxalic  acid  after  subcutaneous 
injections  of  sodium  saccharate.  The  increase,  however,  is  very 
slight.  Pohl,3  after  giving  five  grams  of  sodium  saccharate  to  a 
small  dog,  was  unable  to  detect  any  secondary  oxidation  products. 
The  urine  was  reported  to  contain  neither  saccharic  acid  nor  an 
increase  in  oxalic  acid.  On  the  other  hand,  Schott4  in  experi- 
ments on  rabbits  and  dogs  has  recently  found  that  saccharic  acid 
is  not  oxidized  in  the  body,  but  is  excreted  unchanged  in  the  urine. 

After  giving  large  doses  of  sugar,  an  increase  in  urinary  oxalates 
has  been  observed  in  dogs  by  Baldwin,5 and  in  rabbits  by  P.  Mayer6 
and  Hildebrandt. 7  Baldwin  interprets  the  oxaluria  as  due  to  a 
special  kind  of  gastric  fermentation,  while  Hildebrandt  and  Mayer 
consider  it  as  indicative  of  an  oxidation  of  sugar  through  the  oxalic 
acid  stage.  But,  as  Magnus-Levy8  has  pointed  out,  the  extremely 
large  doses  of  sugar  (40  grams  for  a  rabbit)  may  exert  a  toxic  action 
leading  to  tissue  disintegration  which  latter  might  give  rise  to  the 
oxalic  acid.  In  fact  there  is  considerable  evidence  indicating  the 
possibility  of  oxalic  acid  being  an  intermediate  product  in  the 
metabolism  of  the  protein  substance,  or  of  the  purines. 

As  early  as  1875,  Furbringer9  claimed  that  diabetics  excrete 
excessive  amounts  of  oxalic  acid,  owing  to  decreased  oxidative 

thierfelder:  Zeitschr.  f.  physiol.  Chem.,  xi,  pp.  388-409,  1887. 
2 Mayer:  loc.  cit. 

3Pohl:  Arch.  f.  exp.  Path.  u.  Pharm.,  xxxvii,  pp.  413-25,  1896. 

4 Schott:  Ibid.,  lxv,  pp.  35-7,  1911. 

5Baldwin:  Journ.  of  Exp.  Med.,  v,  pp.  27^L6,  1900-01. 

6 Mayer:  loc.  cit. 

7 Hildebrandt:  Zeitschr.  f.  physiol.  Chem.,  xxxv,  pp.  141-52,  1902. 
8 Magnus-Levy :  Oppenheimer' s  Handb.  d.  Biochem.,  iv,  Part  i,  p.  331, 
1909. 

9 Furbringer:  Deutsch.  Arch.  f.  klin.  Med.,  xvi,  pp.  499-526,  1875; 
xviii,  pp.  143-92,  1876. 


126 


Metabolism  of  Mucic  Acid 


functions,  but  Luzzatto1  has  been  unable  to  observe  any  rise  in 
the  elimination.  Indeed,  it  may  be  said  that  there  is  no.  convinc- 
ing evidence  supporting  the  assumption  that  glycuronic,  saccharic, 
mucic,  and  oxalic  acids  are  produced  under  physiological  condi- 
tions by  an  incomplete  combustion  of  carbohydrates. 

Considering,  however,  the  paucity  of  data  in  regard  to  the  behav- 
ior and  fate  of  mucic  acid  when  introduced  into  the  animal  organ- 
ism, and  since  if  the  unsplit-chain  oxidation  theory  is  correct  one 
would  expect  mucic  acid  to  result  from  the  combustion  of  galac- 
tose, just  as  saccharic  acid  would  result  from  the  oxidation  of 
dextrose,  it  seemed  desirable  to  conduct  experiments  with  mucic 
acid  similar  to  those  of  P.  Mayer  with  glycuronic  and  saccharic 
acids. 

Baumgarten2  administered  20  to  50  gram  doses  of  the  potas- 
sium salt  of  mucic  acid  per  os,  to  normal  and  diabetic  dogs  and  men, 
anol  was  unable  to  recover  any  of  the  acid  in  the  urine.  From 
these  experiments  he  concluded  that  mucic  acid,  in  such  quanti- 
ties, is  readily  and  completely  oxidized  in  the  body.  The  urines 
were  not  analyzed  for  possible  oxidation  products.  Baer  and  Blum3 
found  that  the  feeding  of  mucic  acid  is  without  effect  on  the  keton- 
uria  induced  by  phlorhizin  diabetes.  This  indicates  that  if  mucic 
acid  is  oxidized  in  the  body,  it  is  unable  to  replace  carbohydrate 
as  an  acidosis-inhibiting  agent.  Baer  and  Blum  also  observed 
that  mucic  acid  exerted  a  toxic  action,  resulting  in  the  death  of 
their  animals.  It  is  probable  that  this  was  due  to  the  presence 
of  impurities  in  their  preparation.  The  crude  product  always 
contains  toxic  nitro-compounds,  which  are  removed  only  after 
repeated  recrystallization. 

In  the  present  investigation  mucic  acid  was  fed  to  rabbits  and 
dogs,  and  the  urines  examined  for  mucic  and  oxalic  acids.  In  the 
earlier  experiments  the  mucic  acid  used  was  made  from  lactose  by 
oxidation  with  nitric  acid,  according  to  the  method  of  Kent  and 
Tollens4  and  subsequently  purified  by  recrystallization  until  the 


luzzatto:  Salkowski's  Festschrift,  pp.  239-52,  1904. 
2 Baumgarten:  Zeitschr.  f.  exp.  Path.  u.  Therap.,  ii,  pp.  53-74,  1906. 
"Baer  and  Blum:  Arch.  f.  exp.  Path.  u.  Pharm.,  lxv,  pp.  1-32,  1911. 
4 Kent  and  Tollens:  Liebig's  Annalen,  ccxxvii,  pp.  221-32,  1885. 


William  C.  Rose 


127 


resulting  product  was  perfectly  non-toxic  and  melted  at  213°  C. 
Later  Kahlbaum's  "  Schleimsaure"  was  used  with  equally  satis- 
factory results. 

EXPEKIMENTAL  PART. 1 

Methods. 

The  oxalic  acid  was  estimated  according  to  the  method  of  Sal- 
kowski2  which,  from  the  result  of  comparative  analyses  made 
recently  by  MacLean,3  seems  to  be  less  subject  to  error  than  the 
more  commonly  used  method  of  Autenrieth  and  Barth.4 

It  was  impossible  to  determine  the  mucic  acid  excretion  quanti- 
tatively, on  account  of  want  of  an  adequate  method;  but  quali- 
tative tests  were  made  by  oxidizing  the  urines  with  concentrated 
nitric  acid,  as  Bauer5  has  proposed  in  testing  for  galactose. 

For  this  purpose,  100  cc. -portions  of  the  urine  were  introduced  into 
beakers,  each  portion  treated  with  20  cc.  of  concentrated  nitric  acid  (sp. 
gr.,  1.4),  and  evaporated  on  the  water-bath  to  a  volume  of  approximately 
20  cc.6  The  contents  of  the  several  beakers  were  then  combined,  trans- 
ferred to  a  small  crystallizing  dish,  further  evaporated,  and  allowed  to 
stand  in  a  cool  place  over  night.  The  pure  white  crystals  were  collected 
on  a  small  filter  paper,  washed  two  or  three  times  with  water  and  alcohol, 
dried  in  a  dessicator,  and  identified  by  the  melting  point.  Preliminary 
tests  showed  that  0.2  gram  of  mucic  acid,  when  added  to  100  cc.  of  urine, 
could  be  readily  detected  by  this  method.  Five-tenths  of  a  gram  could 
be  detected  with  the  greatest  ease.  In  one  test  where  the  latter  quantity 
was  added  to  100  cc.  of  urine,  0.26  gram,  or  over  50  per  cent,  was  recovered 
after  oxidation. 


1This  investigation  was  undertaken  at  the  suggestion  of  Professor 
Lafayette  B.  Mendel,  and  the  experimental  data  are  taken  from  the  thesis 
presented  by  the  author  for  the  degree  of  Doctor  of  Philosophy,  Yale 
University,  1911. 

2Salkowski:  Zeitschr.  f.  physiol.  Chem.,  xxix,  pp.  437-60,  1900. 

3 MacLean:  Ibid.,  lx,  pp.  20-24,  1909. 

4Autenrieth  and  Barth:  Ibid.,  xxxv,  pp.  327-42,  1902. 

6Bauer:  Ibid.,  li,  pp.  158-66,  1907. 

6  A  flocky,  gelatinous,  precipitate  of  silica  usually  separated  when  rab- 
bits' urine  was  oxidized  with  nitric  acid.  This  was  removed  by  filtra- 
tion before  crystallizing  the  mucic  acid. 


128 


Metabolism  of  Mucic  Acid 


In  testing  for  mucic  acid  in  the  feces,  the  following  procedure  was  em- 
ployed. The  total  fecal  excretion  for  the  period  was  rubbed  up  with  water, 
the  mixture  made  distinctly  alkaline  with  sodium  hydroxide,  heated 
below  boiling  for  five  to  ten  minutes,  and  strained  through  absorbent 
gauze.  The  residue  was  in  this  manner  extracted  three  times  with  water 
and  alkali,  the  extracts  combined,  divided  into  100  cc. -portions,  and  oxi- 
dized with  nitric  acid,  20-30  cc.  of  concentrated  nitric  acid  being  used  for 
each  100  cc. -portion  of  the  extract.  The  total  volume  of  the  extract  was 
800-1000  cc.  for  the  excreta  of  a  dog  for  two-day  periods.  In  testing  the 
delicacy  of  this  method,  it  was  found  that  half  a  gram  of  mucic  acid  when 
added  to  the  day's  fecal  discharge  of  a  dog,  could  be  almost  quantita- 
tively recovered. 

The  urines  of  the  experimental  animals  were  collected  in  periods 
of  forty-eight  hours.  In  the  rabbits,  the  complete  two  days'  excre- 
tion was  obtained  by  squeezing  out  the  bladders.  With  the  dogs, 
no  attempt  was  made  to  mark  off  the  periods  sharply. 

Usually  the  animals  were  kept  upon  constant  diets  throughout 
the  experiments,  but  occasionally  they  refused  to  eat  as  much 
during  the  experimental  period  as  they  did  during  the  fore  period. 
In  such  cases  a  corresponding  reduction  was  made  in  the  diet  of 
the  after  period,  so  that  in  every  case  the  urinary  findings  during 
the  period  of  mucic  acid  feeding  are  comparable  with  those  result- 
ing from  the  same  diet  without  the  acid.  The  attempt  to  give 
the  acid  in  the  form  of  the  neutral  sodium  salt  almost  invariably 
evoked  diarrhoea,  so  that  this  method  of  administration  was  aban- 
doned. It  is  probable  that  the  large  amount  of  sodium  chloride 
formed  in  the  stomach,  through  the  action  of  the  hydrochloric  acid 
upon  the  sodium  mucate,  was  responsible  for  the  diarrhoea,  by 
inducing  a  large  secretion  of  water  into  the  intestine.  This  ex- 
planation is  rendered  more  probable  inasmuch  as  doses  of  5 
grams  of  sodium  chloride  produce  diarrhoea  in  rabbits.  Hence, 
the  rabbits  received  the  free  mucic  acid  suspended  in  water,  by 
the  stomach  sound.  With  the  dogs,. the  acid  was  mixed  with  the 
finely  hashed  meat  and  dog  biscuit  of  the  diet,  and  fed  at  regular 
intervals. 

Rabbits. 

The  results  of  the  nine  series  of  experiments  on  rabbits  are  sum- 
marized in  Tables  I  to  III.  In  animals  1,  2,  3,  and  4,  the  urine 
was  not  tested  for  mucic  acid,  the  oxalic  acid  excretion  alone  being 


William  C.  Rose  129 


TABLE  I. 


RABBIT 

DURA- 
TION OF 
PERIOD 

VOLUME 
URINE 

MUCIC  ACID 
GIVEN 

OXALIC 

ACID 
OUTPUT 

DIET,  NOTES,  ETC. 

days 

CC. 

gms. 

mgms. 

2 

132 

0 

6.2 

100  gms.  carrots  per 
day. 

100  gms.   carrots  per 

(1)    1.6  kilos  < 

day.  Mucic  acid  neu- 

2 

100 

10 

13.4 

ijl  clllZiCU.  W 1  \jUL  lx  avll, 

and  given    in  two 
doses,  8  hrs.  apart. 

2 

200 

0 

7.5 

100  gms.   carrots  per 
day. 

2 

420 

0 

10.2 

250  gms.   carrots  per 
day. 

250  gms.  carrots  per 

(2)    2.1  kilos  - 

2 

480 

10 

14.1 

day.     Free  Mucic 
acid  given  in  5  gm. 

UUBCO,    £rt   Ulo.  dJJdl  0. 

2 

320 

0 

9.0 

250  gms.  carrots  per 
day. 

'200  gms.  carrots,  25 

2 

365 

0 

11.7 

gms.  corn,  and  100  cc. 
water  per  day. 

200  gms.  carrots,  25 
gms.  corn,  and  100 

(3)    2.0  kilos  < 

2 

360 

10 

18.6 

cc.  water  per  day. 
Mucic  acid  given  in 
two  equal  doses,  24 
^    hrs.  apart. 

200  gms.   carrots,  25 

2 

375 

0 

5.9 

gms.     corn,  and  100 
^cc.  water  per  day. 

(4)    2.2  kilos  | 

500  gms.  carrots  and 

2 

525 

0 

9.1 

200  cc.  water  during 
period. 

130  Metabolism  of  Mucic  Acid 


TABLE  I. — Continued. 


BABBIT 

DURA- 
TION OF 
PERIOD 

VOLUME 
URINE 

MUCIC  ACID 
GIVEN 

OXALIC 

ACID 
OUTPUT 

DIET,  NOTES,  ETC. 

days. 

CC. 

gms. 

mgms. 

Ate  only  355  gms.  car- 

rots during  period. 

Given  200  cc.  water. 

2 

255 

15* 

10.2 

Mucic  acid  given  in 

two   doses  :-lst,  10 

(4)   2.2  kilos  • 

gms.;  2nd,  5 gms.,  24 

L    hrs.  apart. 

'355  gms.  carrots  and 

2 

350 

0 

7.7 

1 

200  cc.  water  dur- 

ing period. 

*On  evaporating  the  urine  for  the  oxalic  acid  estimation,  0.1  gm.  mucic  acid  separated. 
M.P.=213°C. 


determined.  In  these  experiments  as  in  those  on  animals  6,  7,  8, 
and  £),  there  was  only  a  slight  increase  in  the  oxalic  acid  output 
during  the  experimental  periods,  over  that  of  the  normal  periods. 
The  greatest  percentage  increase  was  noted  in  rabbit  7.  Here, 
the  output  was  1.9  milligrams  for  the  fore  periods,  7.2  milligrams 
after  receiving  10  grams,  and  9.8  milligrams  after  receiving  20 
grams  of  mucic  acid.  Even  here  the  actual  increase  is  extremely 
small — far  less  than  one  would  expect  if  mucic  acid  were  normally 
converted  into  oxalic  acid  in  process  of  oxidation. 

Part  of  the  mucic  acid  was  recovered  unchanged  in  the  urine  in 
every  case  after  giving  doses  as  large  as  15  grams,  with  the  possible 
exception  of  rabbit  7.  Here  after  20  grams  given  in  two  doses  of 
10  grams  each,  only  a  trace  of  an  organic  precipitate  was  obtained, 
having  the  crystalline  form  of  mucic  acid. 

In  the  periods  when  10  grams  of  mucic  acid  were  given,  the  unal- 
tered acid  was  recovered  only  once  in  amount  large  enough  to 
identify,  and  here  the  total  quantity  given  was  introduced  in  one 
dose.  When  the  acid  was  given  in  two  doses  of  5  grams  each,  24 
hours  apart,  only  a  very  small  amount  or  none  could  be  detected 
in  the  urine. 

From  the  urine  of  animal  7,  a  trace  of  unaltered  acid  was  recov- 
ered after  a  dose  of  20  grams,  while  the  urine  of  rabbit  8,  a  larger 
animal,  yielded  0.2  gram  of  acid  after  a  dose  of  10  grams.  Whether 


William  C.  Rose  131 


TABLE  II. 


BABBIT 

DURA- 
TION OF 
PERIOD 

VOLUME 
URINE 

MUCIC 
ACID 
GIVEN 

MUCIC  ACID 
RECOVERED 

OXALIC 

ACID 
OUTPUT 

DIET,  NOTES,  ETC. 

days 

2 

CC. 

230 

gms. 

0 

gms. 

0 

mgms. 

Constant  diet 
of  150  gms.  car- 
rots   npr  rlav 

xyjvkj          v^i.  viewy 

throughout  the 
PYnprimpnt. 

(5)    1.3  kilos  I 

2 

340 

15 

0.5 
(M.P.= 
209°) 

fist  day,  5  gms. 
|  mucic  acid. 
1  2d  day,  10  gms. 
[  free  mucic  acid. 

2 

345 

0 

0 

2 

250 

0 

0 

2 

225 

0 

0 

f400  gms.  carrots 
I  and 50 gms.  coin 
[  during  period. 

2 

300 

10 

0 

Ate  only  300  gms. 
carrots  and  50 
gms.  corn  dur- 
■   ing  period.  Mu- 
cic acid  given  in 
2  equal  doses, 
24  hrs.  apart. 

(6)   2.0  kilos.... 

2 

195 

0 

0 

f300  gms.  carrots 
<  and 50 gms.  corn 
[  during  period. 

2 

200 

15 

0.15 
(M.P.= 
209°) 

3.9 

Same  diet  as  in 
preceding  pe- 
riod.   1st  day, 
•   10    gms.  free 
mucic  acid.  2d 
day,  5  gms.  free 
mucic  acid. 

2 

250 

0 

0 

3.4 

fSame  diet  as  in 
\preceding  period. 

132  Metabolism  of  Mucic  Acid 


TABLE  II— Continued. 


BABBIT 

DURA- 
TION OF 
PERIOD 

VOLUME 
URINE 

MUCIC 
ACID 
GIVEN 

MUCIC  ACID 
RECOVERED 

OXALIC 

ACID 
OUTPUT 

DIET,  NOTES,  ETC. 

days 

CC. 

gms. 

gms. 

mgms. 

\  Constant  diet  of 

2 

260 

0 

0 

1.9 

1 

200  gms.  carrots 
per day through- 
ouT/  experiment. 

Mucic  acid  given 

(7)    1.6  kilos  ...  ■ 

2 

350 

10 

0 

7.2 

! 

in    two  equal 
doses,   24  hrs. 
apart. 

1st  day,  10  gms. 

2 

450 

20 

Trace* 

9.8 

1 

free  mucic  acid. 
2d  day,  5  gms. 
free  mucic  acid. 

2 

450 

0 

0 

5.4 

*  Very  small  precipitate  was  obtained  which  was  identified  as  organic  matter. 


this  was  due  to  individual  variations  in  the  ability  to  oxidize  mucic 
acid,  or  to  differences  in  rate  and  degree  of  absorption,  could  not 
be  determined. 

Dogs. 

Two  experiments  were  made  upon  dogs.  Dog  10  (Table  IV) 
was  a  small  animal,  and  excreted  relatively  large  amounts  of  the 
ingested  mucic  acid.  Throughout  the  experiment  he  was  kept 
upon  a  constant  diet,  consisting  of  100  grams  of  lean  meat,  40 
grams  of  cracker  meal,  and  25  grams  of  lard  per  day.  While  the 
urines  did  not  represent  exactly  48  hour  periods,  the  oxalic  acid 
excretion  agrees  very  well  with  the  results  obtained  in  rabbits. 
Here  also  the  actual  increase  during  the  mucic  acid  periods  is 
slight  and  insignificant. 

In  animal  11,  a  short  experiment  was  made  to  determine  whether 
or  not  any  of  the  mucic  acid  failed  to  be  absorbed,  and  was  excreted 
in  the  feces.  The  dog  was  fed  15p  grams  of  meat,  100  grams  of 
dog  biscuit,  and  30  grams  of  lard  per  day.    The  feces  were  marked 


William  C.  Rose  133 


TABLE  III. 


RABBIT 

DURA- 
TION OF 
PERIOD 

VOLUME 
URINE 

MUC1C 
ACID 
GIVEN 

MUCIC  ACID 
RECOVERED 

OXALIC 

ACID 
OUTPUT 

DIET,  NOTES,  ETC. 

days 

2 

CC. 

210 

gms. 

0 

gms. 

0 

mgms. 

5.9 

{ 

'400  gms.  carrots 
during  period. 

2 

170 

10 

0.2 
(M.P.= 
212°) 

6.9 

Ate  only  350  gms. 
carrots  during 
period.  Mucic 
acid  given  in 
one  dose. 

(8)    1.8  kilos....  • 

2 

300 

0 

0 

7.1 

Same  diet  as  in 
preceding  peri- 
k  od. 

2 

350 

20 

0.5 
(M.P.= 
213°) 

8.2 

Same  diet  as  in 
preceding  peri- 
od. Mucic  acid 
given  in  two 
equal  doses,  24 
hrs.  apart. 

0 

450 

0 

0 

7.9 

rSame  diet  as  in 
preceding  peri- 
od. 

• 

2 

275 

0 

0 

5.6 

Constant  diet  of 
150  gms.  car- 
rots per  day 
throughout  ex- 
periment. 

(9)    1.4  kilos 

2 

350 

•  10 

Trace* 

8.5 

< 

Mucic  acid  given 
in  two  equal 
doses,  24  hrs . 
apart. 

2 

350 

0 

0 

6.4 

2 

400 

15 

0.5 
(M.P.= 
212°) 

10.0 

1st  day,  10  gms. 
mucic  acid.  2d 
day,  5  gms. 
mucic  acid. 

2 

300 

0 

0 

6.7 

*Organic  matter.   Melting  point  not  determined. 


134 


Metabolism  of  Mucic  Acid 


TABLE  IV. 
Dog  10;  6.8  kilos. 


DURATION 
OF 
PERIOD 

VOLUME 
URINE 

SPECIFIC 
GRAV- 
ITY 

REAC- 
TION TO 
LITMUS 

MUCIC 
ACID 
GIVEN 

MUCIC  ACID 
RECOVERED 

OXALIC 

ACID 
OUTPUT 

NOTES 

days 

cc. 

gms. 

gms. 

mgms. 

2 

500 

1.020 

acid 

0 

0 

13.4 

z 

1  HQ9 

acid 

n 

u 

u 

IRA 
ID .  D 

2 

300 

1.032 

acid 

10 

0.3 
(M.P.= 
210°) 

1.3 

25.1 

fm*.  • 

Mucic  acid  was 
I  given  in  one  dose. 

fMucic  acid  was 

2 

410 

1.037 

acid 

20 

(M.P.= 
210°) 

30.5 

\  given  in  two  equal 
[  doses, 24  hrs. apart. 

2 

275 

1.027 

acid 

0 

0 

7.8 

TABLE  V. 
Dog  11;  13.2  kilos. 


>URATION 

OF 
PERIOD 

VOLUME 
URINE 

SPECIFIC 
GRAV- 
ITY 

REACTION  TO 
LITMUS 

MUCIC 
ACID 
GIVEN 

MUCIC 
ACID 
RECOV- 
EREDIN 
URINE 

MUCIC 
ACID 
RECOV- 
ERED IN 

FECES 

NOTES 

days 

CC. 

gms. 

gms. 

gms. 

1 

176 

1.050 

alkaline 

0 

0 

0 

fMucic    acid  given 

2 

300 

1.046 

acid 

20 

0 

0 

<  in  two  equal  doses, 

[  24  hrs.  apart. 

250 

1.027 

acid 

0 

0 

0 

1st  day  of  period. 

170 

1.025 

alkaline 

0 

0 

0 

2nd  day  of  period. 

off  into  two-day  periods  with  lamp-black,  and  analyzed  for  mucic 
acid.  The  results  are  shown  in  Table  V.  After  a  dose  of  20  grams, 
not  a  trace  of  the  acid  was  recovered  in  the  feces.  Apparently, 
mucic  acid  is  readily  absorbed  even  when  given  in  the  free  state. 
None  was  recovered  in  the  urine  of  this  animal  after  the  mucic 
acid  ingestion.    The  urine,  which  was  alkaline  to  litmus  for  some 


William  C.  Rose 


135 


unknown  reason  during  the  fore  period  and  last  day  of  the  after 
period,  was  rendered  strongly  acid  for  three  days  after  giving  the 
mucic  acid.  Whether  or  not  the  increased  urinary  acidity  was 
due  to  a  slight  rise  in  the  output  of  oxalic  acid,  or  to  the  presence 
of  traces  of  mucic  acid  too  small  to  be  detected  by  the  method,  was 
not  determined. 

Since  rabbits  and  dogs — at  least  dog  10 — excrete  unaltered 
mucic  acid  in  the  urine  after  20-gram  doses,  it  seems  scarcely  prob- 
able that  mucic  acid  is  an  intermediary  product  in  the  combustion 
of  galactose  and  galactose-yielding  carbohydrates.  To  further 
test  this  hypothesis,  however,  a  series  of  experiments  were  made  in 
which  animals  received  the  calculated  amount  of  lactose  (or  gal- 
actose) necessary  to  yield  20  grams  of  mucic  acid  in  the  organism, 
and  the  urines  were  analyzed  for  mucic  acid.    The  results  follow: 

Experiment  1.  A  rabbit  weighing  2300  grams,  received  by  the  stom- 
ach-tube 35  grams  of  lactose,  in  two  equal  doses,  twenty-four  hours 
apart.  Thirty-five  grams  of  lactose,  if  oxidized  through  the  mucic  acid 
stage,  should  yield  21.4  grams  of  mucic  acid.  The  animal  was  well  fed 
on  carrots  and  oats,  and  the  urine  collected  for  sixty  hours.  The  urine 
contained  no  sugar  or  mucic  acid. 

Experiment  2.  A  rabbit  weighing  1800  grams,  received  35  grams  of 
lactose  in  two  equal  doses,  twenty-four  hours  apart.  The  urine  excreted 
during  the  following  fifty  hours  contained  no  sugar  or  mucic  acid. 

Experiment  3.  A  rabbit  weighing  2020  grams,  received  two  doses 
of  lactose,  17.5  grams  each,  twenty-four  hours  apart.  The  urine  of  the 
following  fifty  hours  contained  no  sugar  or  mucic  acid. 

Experiment  4.  A  rabbit  weighing  2200  grams,  received  8.6  grams  of 
galactose.  After  an  interval  of  twenty-four  hours,  a  second  dose  of  the 
same  amount  was  administered.  The  total  amount  given  (17.2  grams), 
if  oxidized  through  the  mucic  acid  stage,  should  yield  20  grams  of  mucic 
acid .  The  urine  for  the  next  sixty  hours,  contained  a  minute  trace  of  sugar. 
No  mucic  acid  was  obtained  after  oxidizing  with  nitric  acid.  The  amount 
of  sugar  present,  if  galactose,  was  too  small  to  be  detected  by  the  Bauer 
method. 

Experiment  5.  A  dog  weighing  5.4  kilos,  was  given  35  grams  of  lac- 
tose in  two  equal  doses  mixed  in  with  the  food.  The  urine  of  the  following 
fifty  hours  gave  a  slight  reduction  of  Benedict's  solution,  no  test  with  Feh- 
ling's  solution,  and  yielded  no  mucic  acid  on  oxidation  with  nitric  acid. 

Experiment  6.  The  same  animal  used  in  Experiment  5,  received  35 
grams  of  lactose  in  one  dose.  The  urine  of  the  following  forty-eight  hours 
gave  a  distinct  reduction  of  Fehling's  solution.  The  sugar  was  removed 
by  fermenting  with  ordinary  yeast  (showing  that  the  sugar  was  not  lac- 
tose), the  yeast  filtered  off,  and  the  filtrate  oxidized  with  nitric  acid.  No 
mucic  acid  was  obtained. 


136 


Metabolism  of  Mucic  Acid 


Experiment  7.  A  dog  weighing  6.2  kilos,  received  35  grams  of  lac- 
tose in  one  dose.  The  urine  of  the  following  forty-eight  hours  contained 
a  minute  trace  of  sugar.  Direct  oxidation  with  nitric  acid  yielded  no 
mucic  acid. 

GENERAL  DISCUSSION. 

The  results  of  the  mucic  acid  feeding  experiments,  particularly 
those  upon  dogs,  are  not  in  accord  with  the  findings  of  Baumgarten1 
This  investigator  was  unable  to  detect  mucic  acid  in  the  urine  of  a 
medium  size  dog,  after  giving  20  grams,  and  in  the  urines  of  nor- 
mal and  diabetic  men  after  giving  50  grams  of  the  acid.  The 
failure  to  detect  mucic  acid  in  the  urine  in  any  case,  was  probably 
due,  at  least  in  part,  to  the  inadequate  method  employed,  which 
consisted  in  evaporating  the  urine  made  alkaline  with  sodium 
hydroxide  to  dryness,  extracting  the  residue  with  hot  absolute 
alcohol  to  remove  the  urea,  and  preparing  the  double  hydrazine 
compound  of  mucic  acid,  C4H4(OH)4.  (CO .  N2H2.C6H5)2,  by  boil- 
ing on  the  water-bath  with  sodium  acetate  and  phenylhydrazine- 
hydrochloride.  Billow,2  who  first  prepared  the  compound,  heated 
the  mucic  acid  and  phenylhydrazine  on  an  oil-bath,  at  a  tempera- 
ture of  120°  to  140°  C.  He  states  that  the  reaction  is  not  complete 
until  all  water  has  been  evaporated.  Baumgarten  seems  to  have 
overlooked  these  facts,  although  he  cites  the  paper  of  Biilow  in 
describing  the  method.  It  was  found  impossible  to  obtain  the 
compound  by  Baumgarten's  method  of  heating  on  a  water-bath, 
even  after  the  addition  of  2  grams  of  mucic  acid  to  the  100  cc.  of 
urine  used  in  the  test.  It  seems  scarcely  probable  that  Baum- 
garten would  have  used  the  method  without  first  testing  its  deli- 
cacy, but  if  such  tests  were  made  no  mention  of  the  fact  is  to  be 
found  in  his  paper. 

In  both  the  rabbit  and  dog  experiments,  the  increase  in  oxalic 
acid  excretion  observed  after  giving  mucic  acid  compares  favor- 
ably in  amount  with  that  found  by  Mayer3  after  giving  doses  of 
15  to  20  grams  of  sodium  glycuronate  or  sac  char  ate,  and  interpreted 
by  him  as  indicating  an  oxidation  of  these  substances  to  oxalic 
acid  in  process  of  metabolism.    A  similar  interpretation  is  made 

1  Baumgarten:  loc.  cit. 

2 Biilow:  Liebig's  Annalen,  ccxxxvi,  pp.  194-97,  1886. 
3 Mayer:  loc.  cit. 


William  C.  Rose 


137 


of  the  increased  oxalic  acid  excretion  observed  after  giving  rabbits 
40-gram  doses  of  dextrose,  although  the  largest  actual  increase 
obtained  was  from  1.2  milligrams  before,  to  4.7  milligrams  after 
the  sugar  administration.  It  would  seem  that  the  explanation 
of  this  small  increase  in  oxalic  acid  suggested  by  Magnus-Levy1 
and  already  alluded  to,  is  much  more  probable — namely,  that  the 
abnormally  large  doses  of  foreign  substances  produce  a  slight  dis- 
integration of  body  tissue,  sufficient  to  occasion  a  rise  in  oxalate 
elimination. 

It  is  not  at  all  likely  that  oxalic  acid  is  an  intermediary  product 
in  the  normal  metabolism  of  any  of  the  food-stuffs.  Under  cer- 
tain abnormal  conditions,  at  present  little  understood,  it  arises 
in  the  body,  and  is  immediately  excreted.  This  conception  would 
seem  to  be  indicated  from  the  results  of  feeding  experiments  with 
oxalic  acid.  The  inability  of  the  organism  to  oxidize  it,  when  intro- 
duced per  os  or  subcutaneouly,  is  conclusively  shown  by  the  re- 
searches of  Gaglio2  and  Pohl.3  Recently  the  latter  investigator,4 
has  corroborated  his  former  work,  having  quantitatively  recovered 
in  the  urine  all  the  oxalic  acid  introduced  into  the  organism.  At 
any  rate,  no  evidence  has  been  obtained  from  feeding  experiments 
with  mucic,  saccharic,  and  glycuronic  acids,  that  oxalic  acid  is  a 
normal  intermediary  product  of  carbohydrate  metabolism. 

Again,  from  the  results  of  the  lactose  feeding  experiments,  the 
oxidation  of  the  end  carbon  atoms  of  the  sugar  molecule  without 
preliminary  cleavage  seems  unlikely.  In  all  experiments  with 
the  exception  of  experiment  6,  (in  which  the  lactose  was  given  in 
one  dose,)  the  sugar  was  completely  utilized  by  the  animals. 
Usually  the  sugar  was  given  in  two  doses,  24  hours  apart,  in  order 
to  make  the  experiments  exactly  comparable  with  the  mucic 
acid  experiments.  Twenty  grams  of  mucic  acid  given  in  this 
manner,  are  not  entirely  oxidized  by  the  body,  but  reappear 
in  part  in  the  urine.  Stoichiometrically  equivalent  amounts  of 
galactose-sugars,  given  under  the  same  conditions,  fail  to  evoke 
the  appearance  of  mucic  acid  in  the  urine.  The  conclusion  is 
obvious — mucic  acid  is  not  a  product  of  the  physiological  metabolism 

Magnus-Levy:  loc.  cit. 

2Gaglio:  Arch.  f.  exp.  Path.  u.  Pharm.,  xxii,  pp.  235-52,  1887. 
3 Pohl:  loc.  cit. 

i Pohl:  Zeitschr.  f.  exp.  Path.  u.  Therap.,  viii,  pp.  308-11,  1910. 


138  Metabolism  of  Mucic  Acid 

of  galactose.  It  may  be  objected,  that  in  the  earlier  experiments 
too  much  acid  was  introduced  into  the  circulation  at  one  time  to 
allow  complete  oxidation,  while  in  the  latter  series  the  change 
from  lactose  to  mucic  acid  was  very  slow,  only  small  amounts 
arising  each  moment.  This  explanation  is,  however,  scarcely 
valid,  inasmuch  as  mucic  acid  is  relatively  insoluble  and  its  absorp- 
tion must  be  very  slow.  Considerable  time  would  be  necessary  for 
the  content  of  the  blood  in  mucic  acid  to  be  appreciably  increased. 
Hence,  there  should  be  ample  time  for  oxidation  to  occur,  before 
the  circulation  becomes  flooded  with  the  acid.  It  is  probable, 
therefore,  that  sugars  are  normally  oxidized  by  some  method 
other  than  that  indicated  by  the  unsplit-chain  theory. 

SUMMARY. 

1.  Mucic  acid,  in  doses  of  10  to  20  grams,  was  not  completely 
oxidized  by  rabbits,  but  was  in  part  excreted  unchanged  in  the 
urine. 

2.  When  given  in  doses  of  20  grams  to  a  medium  sized  dog, 
mucic  acid  was  excreted  unchanged  in  the  urine  in  amounts  large 
enough  to  identify.  The  greater  portion  of  the  acid  was  not  recov- 
ered. 

3.  After  large  doses  of  mucic  acid,  only  a  very  small  increase 
in  oxalic  acid  elimination  occurs  in  rabbits  and  dogs.  This  increase 
is  by  no  means  as  large  as  would  be  expected  if  mucic  acid  were  one 
of  the  precursors  of  oxalic  acid. 

4.  Rabbits  and  dogs  receiving  the  amounts  of  galactose  and 
lactose  stoichiometrically  equivalent  to  20  grams  of  mucic  acid, 
excrete  no  mucic  acid  in  the  urine.  Obviously,  therefore,  mucic 
acid  is  not  an  intermediary  product  in  the  metabolism  of  galactose- 
yielding  sugars. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  X,  No.  3,  1011 


EXPERIMENTAL  STUDIES  ON  CREATINE  AND 
CREATININE.1 

I.    THE  ROLE  OF  THE  CARBOHYDRATES  IN  CREATINE- 
CREATININE  METABOLISM. 

By  LAFAYETTE  B.  MENDEL  and  WILLIAM  C.  ROSE. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  August  14,  1911.) 
INTRODUCTORY. 

Despite  the  enormous  amount  of  data  that  have  been  accumu- 
lated in  recent  years  in  regard  to  the  occurrence  and  excretion 
of  creatine  and  creatinine,  our  knowledge  of  the  significance 
of  these  substances  is  still  far  from  adequate.  The  incidence 
of  the  excretion  of  creatinine  (cf.  Mendel,  '09  and  Myers,  '10), 
is  perhaps  better  understood  than  that  of  creatine.  Folin  ('05a, 
'05b,  '05c),  van  Hoogenhuyze  and  Verploegh  ('05),  Klercker  ('06), 
Closson  ('06),  and  Shaffer  ('08b)  have  shown  that  the  daily 
output  of  creatinine,  on  a  meat-free  diet,  is  remarkably  con- 
stant for  the  same  individual,  and  is  independent  of  the  total 
nitrogen  and  volume  of  the  urine.  This  constancy  in  the  excre- 
tion of  creatinine  indicates  that  it  is  of  endogenous  origin,  and 
as  Folin  ('05b)  says,  is  an  "  index  of  a  certain  kind  of  protein- 
metabolism  occurring  daily  in  any  given  individual."  Folin  ('06) 
further  believes  that  the  creatinine  of  the  urine  has  no  connec- 
tion with  the  muscle  creatine,  as  the  latter  is  converted  into  creat- 
inine only  with  great  difficulty. 

1A  preliminary  report  of  these  studies  was  presented  to  the  Society  for 
Experimental  Biology  and  Medicine,  May  17,  1911. 

213 


2  14         Creatine  and  Creatinine  Metabolism 


The  excretion  of  creatinine  in  disease  frequently  undergoes 
marked  variation.1  Mellanby  ('07)  found  the  creatinine-coef- 
ficient  to  be  very  low  in  diseases  of  the  liver.  This  was  partic- 
ularly true  of  individuals  with  hepatic  carcinoma.  The  urines 
of  such  patients  contained  large  amounts  of  creatine.  Mellanby 
('08)  believes  that  the  liver  is  intimately  connected  with  the 
production  and  excretion  of  creatinine.  He  suggests  that  the 
liver  is  continuously  forming  it  from  substances  brought  from 
other  organs  by  the  blood.  In  the  developing  muscle,  this  creat- 
inine is  transformed  into  creatine,  and  stored  until  the  mus- 
cles have  reached  a  certain  saturation  point.  After  this  point 
has  been  reached,  creatinine  is  continuously  excreted. 

Both  creatine  and  creatinine  are  excreted  in  the  urine  of  dogs 
with  Eck  fistulas,  according  to  Salaskin  and  Zaleski  ('00),  Lon- 
don and  Boljarski  ('09),  and  Foster  and  Fisher  ('11).  London 
and  Boljarski  found  that  the  administration  of  creatinine  to  such 
animals  did  not  increase  urinary  creatinine.  Results  contra- 
dictory to  these  were  obtained  by  Foster  and  Fisher,  who  report 
that  the  ingestion  of  creatinine  increases  the  creatinine  output 
in  the  urine.  The  results  of  both  investigations  are  in  accord  in 
finding  that  the  feeding  of  creatine  caused  no  increase  in  creatine 
excretion,  but  was  followed  by  a  slight  rise  in  eliminated  creat- 
inine. 

The  observations  of  van  Hoogenhuyze  and  Verploegh  ('05) 
and  Shaffer  ('08a)  as  well  as  of  certain  earlier  investigators, 
indicate  that  increased  or  decreased  muscular  activity,  with 
adequate  food,  has  per  se  no  effect  on  the  excretion  of  creatinine. 

Creatine  is  never  normally  present  in  the  urine  of  adult 
mammals,  unless  creatine  is  taken  in  with  the  food.  As  early, 
however,  as  1868  Meissner  ('68)  found  that  creatine  almost  en- 
tirely replaces  creatinine  in  the  urine  of  birds.  These  observa- 
tions were  subsequently  verified  by  Paton  ('09-' 10)  and  Voegtlin 
and  Towles  ('11). 

In  mammals,  creatine  is  excreted  in  the  urine  during  inanition. 
This  was  first  observed  by  Benedict  ('07),  and  later  verified  in 

1  For  the  literature  on  the  excretion  of  creatinine  in  various  pathological 
conditions,  see  Leathes  ('06-07),  Spriggs  ('07a,  '07b),  Benedict  and  Myers 
('07a),  Forschbach  ('08),  Shaffer  ('08b),  and  Levene  and  Kristeller  ('09). 


Lafayette  B.  Mendel  and  William  C.  Rose  215 


collaboration  with  Diefendorf  (Benedict  and  Diefendorf,  '07)  on  a 
fasting  woman.  At  about  the  same  time,  but  independently, 
Cathcart  ('07a,  '07b)  noted  the  excretion  of  creatine  in  starvation. 
Recently  this  author  (Cathcart,  '09)  has  published  the  results  of 
experiments  made  upon  himself  and  others  in  which  he  reports 
that  creatine  is  found  in  the  urine,  in  relatively  large  amounts, 
after  fasting  periods  of  forty  hours.  In  starvation  experiments 
made  upon  rabbits,  Dorner  ('07)  noted  the  elimination  of  crea- 
tine. Similar  results  were  obtained  in  dogs  by  Underhill  and 
Kleiner  ('08),  Richards  and  Wallace  ('08),  and  Howe  and  Hawk 

Cii). 

Pathologically,  creatine  occurs  in  a  variety  of  conditions. 
Benedict  and  Myers  ('07b)  found  it  in  the  urines  of  a  large  num- 
ber of  insane  patients,  most  of  whom  were  in  poor  nutritive  con- 
dition, which  probably  accounted  for  its  appearance.  In  con- 
valescence after  typhoid  fever  Foster  ('10)  found  an  elimination 
of  creatine.  Shaffer  ('08b)  observed  creatine  to  be  invariably 
excreted  where  there  was  a  rapid  loss  of  muscle  protein,  such  as 
in  acute  fevers,  in  the  acute  stages  of  exophthalmic  goitre,  in  tumor 
cachexia,  and  in  women  during  the  first  week  post  partum,  when 
the  resolution  of  the  muscular  wall  of  the  uterus  is  proceeding 
most  rapidly.  This  has  also  been  observed  in  dogs  by  Murlin 
('08-'09).  According  to  this  author,  the  creatine  first  appeared 
in  the  urine  two  days  before  parturition,  and  reached  a  maximum 
on  the  fifth  day  after  parturition.  He  suggests  that  the  latter 
date  probably  marks  the  maximum  of  the  involution  process. 
Levene  and  Kristeller  ('09)  noted  the  excretion  of  creatine  in  a 
variety  of  diseases.  The  largest  amounts  were  found  in  the  urine 
of  patients  with  anterior  poliomyelitis  and  muscular  dystrophy. 

In  many  of  these  instances — notably  fevers  and  hepatic  car- 
cinoma— under-nutrition  is  undoubtedly  an  important  contrib- 
uting factor  in  the  production  of  creatine.  In  this  connection 
the  recently  published  paper  of  Underhill  and  Rand  ('10)  is  of 
particular  interest.  These  authors  found  large  amounts  of  crea- 
tine in  the  urines  of  women  with  pernicious  vomiting  of  preg- 
nancy, and  Underhill  suggests  that  the  changes  observed  in  the 
urine  are  induced  by  the  accompanying  inanition.  Evidence 
tending  to  substantiate  this  view  is  furnished  by  the  obser- 
vation that  the  perverted  creatine  metabolism,  as  well  as  the 


2i6         Creatine  and  Creatinine  Metabolism 


metabolism  of  the  other  nitrogenous  constituents  of  the  urine, 
tends  rapidly  to  resume  the  normal  on  the  rectal  administration 
of  dextrose,  without  necessarily  exerting  any  influence  on  the 
pathological  state  of  the  patient.  The  presence  of  carbohydrates 
appears  in  these  experiments  to  be  the  all-important  factor  in 
preventing  the  abnormal  partition  of  urinary  nitrogen  associated 
with  starvation.  In  the  absence  of  sufficient  carbohydrates  the 
energy-yielding  substances  in  the  body  seem  to  be  utilized  with 
great  difficulty.  The  inability  to  oxidize  carbohydrates  may, 
therefore,  explain  the  observations  of  Shaffer  ('08b)  and  Dreib- 
holz  ('08);  and  more  recently  of  Krause  ('10),  Krause  and  Cramer 
('10),  and  Taylor  ('10,  '11),  that  creatine  is  a  constant  product 
of  the  metabolism  of  patients  with  diabetes  mellitus. 

In  consideration,  therefore,  of  such  observations  as  the  above, 
a  series  of  experiments  was  conducted  on  starving  rabbits,  to 
determine  the  influence  of  carbohydrates  on  the  creatine  metab- 
olism during  a  period  of  inanition  unaccompanied  by  any  other 
abnormal  factor. 

From  two  experiments  upon  geese,  Paton  ('09-' 10)  reached  the 
conclusion  that  the  administration  of  glucose  in  fasting  has  no 
specific  action  on  the  excretion  of  creatine.  But  his  experiments 
were  entirely  too  few  in  number,  and  extended  over  too  short 
periods  of  time,  to  render  his  results  conclusive. 

While  the  present  work  was  in  progress,  an  interesting  paper 
appeared  by  Cathcart  ('09),  in  which  he  reports  that  the  creatine 
excretion  induced  by  fasting  is  reduced  to  nil  by  administering  a 
carbohydrate  diet  " practically  nitrogen  and  fat  free;"  whereas 
with  a  fat  diet,  the  amount  of  creatine  excreted  is  increased.  He 
further  states  that  the  addition  of  protein  food  (carbohydrate-free) 
during  the  fat  period,  does  not  markedly  reduce  the  creatine  excre- 
tion. The  experiments  were  made  upon  men,  which  necessarily 
limited  the  duration  of  the  fasting  to  short  periods  (usually  forty 
hours).  The  author  reports  that  this  inanition  always  brought 
about  an  output  of  some  hundred  and  fifty  milligrams  of  creatine, 
which  seems  surprisingly  large  for  a  fast  of  so  short  duration.  Usu- 
ally, several  days  of  starvation  are  necessary  to  induce  the  excre- 
tion of  appreciable  amounts  of  creatine  in  man  (cf.  Benedict  and 
Diefendorf,  '07,  and  Underhill  and  Rand,  '10). 


Lafayette  B.  Mendel  and  William  C.  Rose  217 


The  chief  criticism,  however,  of  Cathcart's  experiments  involves 
the  nature  of  the  carbohydrate  diet  used  to  reduce  the  creatine 
output.  This  consisted  of  tapioca,  sugar,  honey,  corn-flour,  and 
banana  meal,  all  of  which — with  the  exception  of  the  sugar — con- 
tain small  amounts  of  nitrogen.    In  one  experimental  period 


table  1. 
CathcarVs  carbohydrate  diet. 


ARTICLE  OF  FOOD 

AMOUNT  INGESTED 
PER  DAY 

N 

N  INTAKE 

grams 

per  cent 

grams 

Banana  meal  

454 

0.64 

2.80 

Honey  

230 

0.23 

0.53 

3.33 

(Cathcart,  '09,  p.  316)  the  diet  consisted  of  banana  meal  and  honey, 
and  contained  according  to  Cathcart's  own  analyses,  3.33  grams  of 
nitrogen  (see  Table  I).  It  is  true  that  this  is  a  small  nitrogen 
intake,  and  that  for  most  purposes  the  diet  might  be  considered 
" practically  nitrogen  free;"  but,  a  priori,  we  have  no  way  of  know- 
ing whether  the  nitrogen  accompanied  by  the  carbohydrates,  or 
the  carbohydrates  per  se  are  responsible  for  the  reduction  in  crea- 
tine elimination.  Chittenden1  has  shown  that  man  can  live  in 
perfect  health,  and  remain  in  nitrogen  equilibrium,  on  an  intake 
of  5  or  6  grams  of  nitrogen  per  day,  when  sufficient  carbohydrates 
and  fats  are  ingested.  May  it  not,  therefore,  be  possible,  that 
an  intake  of  half  of  the  amount  of  nitrogen  necessary  to  meet  the 
needs  of  the  body  is  capable,  when  accompanied  by  carbohydrates, 
of  exerting  some  influence  on  creatine  elimination  during  starva- 
tion? Certainly  we  have  no  answer  to  this  question  in  the  results 
of  Cathcart.  Hence,  it  was  determined  to  test  the  influence  of 
a  carbohydrate  diet  absolutely  nitrogen-free  on  the  creatine  elim- 
ination in  animals  during  inanition. 

1  Chittenden :  Physiological  Economy  in  Nutrition,  New  York,  1904. 


2lS 


Creatine  and  Creatinine  Metabolism 


EXPERIMENTAL  PART.1 

Methods. 

The  animals  used  in  the  investigation  were  large  rabbits,  pre- 
viously well  fed  on  oats,  cracked  corn  and  carrots.  In  several 
experiments  the  urines  were  analyzed  for  three  or  four  days  before 
the  fasting  periods  were  begun,  in  order  to  determine  whether  or 
not  creatine  is  normally  excreted  by  rabbits  on  a  mixed  diet.  The 
urine  was  always  collected  at  the  end  of  twenty-four  hour  periods, 
unless  otherwise  stated;  the  complete  excretion  for  the  day's  cycle 
being  obtained  by  squeezing  out  the  bladder.  Total  nitrogen  was 
estimated  by  the  Kjeldahl-Gunning  method,  ammonia  nitrogen2 
and  preformed  creatinine3  by  the  Folin  methods,  and  "total 
creatinine,"4  i.e.,  after  conversion  of  creatine  present  to  creatinine, 
by  the  Benedict-Myers  modification  of  the  Folin  method. 

In  describing  the  creatinine  determination  in  rabbits'  urine,  Dorner 
('07)  alludes  to  the  appearance  of  a  flocky  precipitate  after  treating  with 
picric  acid  and  sodium  hydroxide,  the  precipitate  persisting  even  after 
dilution  of  the  mixture  to  500  cc.  He  suggests  filtering  the  solution  before 
making  the  colorimetric  readings.  It  has  been  the  experience  of  the  writer 
that  this  procedure  is  entirely  unnecessary.  The  precipitate  which  is  com- 
posed largely  or  entirely  of  phosphates,  is  so  small  in  amount  that  it  offers 
no  hindrance  to  matching  the  color  accurately  with  the  standard  bichromate 
solution.  Frequently,  in  sufficiently  dilute  urines,  no  visible  precipitate 
appears  at  all. 

Often  a  difference  of  three  or  four  minutes  in  the  time  the  urine  is  allowed 
to  stand  after  the  addition  of  the  picric  acid  and  sodium  hydroxide,  pro- 
duces a  variation  of  a  millimeter  or  more  in  the  colorimetric  reading.  The 
maximum  depth  of  color  is  obtained  in  about  ten  minutes.  Consequently, 
in  all  the  determinations  of  creatine  and  creatinine  recorded  below,  ten 
minutes  were  allowed  for  the  completion  of  the  reaction.  The  solutions 
were  then  diluted  and  the  readings  taken  immediately. 


1  The  experimental  data  are  taken  from  the  thesis  presented  by  William 
C.  Rose  for  the  degree  of  Doctor  of  Philosophy,  Yale  University,  1911. 
2 Folin:    Amer.  Journ.  Physiol.,  xiii,  pp.  45-65,  1905. 
3 Folin:    Zeitschr.  f.  physiol.  Chem.,  xli,  pp.  223-42,  1904. 
4  Benedict  and  Myers:    Amer.  Journ.  Physiol.,  xviii,  pp.  397-405,1907. 


Lafayette  B.  Mendel  and  William  C.  Rose  219 


The  influence  of  inanition  on  the  creatine-creatinine  excretion  in 
rabbits,  with  some  observations  on  the  elimination  of  ammonia. 

Four  rabbits  were  allowed  to  starve  until  death  resulted,  the 
urines  being  analyzed  for  total  nitrogen,  ammonia  nitrogen,  total 
and  preformed  creatinine.  The  analytical  data  are  summarized 
in  Tables  II  to  V.  Throughout  the  experiments  the  animals  were 
given  water  ad  libitum.  Creatine  usually  appeared  in  the  urine 
on  the  second  day,  and  progressively  increased  in  actual  amount 
until  death.  There  was  generally  also  an  increase  in  the  percentage 
of  the  total  nitrogen  present  as  creatine,  but  considerable  varia- 
tion is  noted  owing  to  the  large  increase  in  total  nitrogen.  The 
elimination  of  nitrogen  in  the  form  of  creatinine  is  remarkably  constant. 
Though  slight  fluctations  are  noted — especially  a  tendency  to 
decrease  just  before  death — as  in  rabbits  1  and  3,  the  change  is 
by  no  means  commensurate  with  that  observed  in  the  creatine 
elimination.  The  percentage  of  creatinine-nitrogen  invariably 
decreases  as  inanition  progresses,  on  account  of  the  increased  total 
nitrogen  elimination.  These  results  agree  with  those  of  Dorner 
('07). 

Ammonia  Elimination.  Incidentally  it  is  of  interest  to  note 
the  elimination  of  ammonia  nitrogen.  So  far  as  the  writer  is 
aware,  no  determinations  of  the  ammonia  excretion  in  rabbits  dur- 
ing starvation  have  been  previously  published.  It  was  expected 
that  the  percentage  of  the  total  nitrogen  present  in  the  form 
of  ammonia  would  be  increased,  but  such  is  not  the  case.  With 
the  exception  of  rabbit  1,  where  the  results  are  very  irregular,  all 
animals  excreted  progressively  smaller  percentages  of  their  nitro- 
gen as  ammonia.  Usually,  there  is  a  tendency  for  the  absolute 
amount  to  slightly  increase  before  death,  but  this  increase  does 
not  keep  pace  with  the  increase  in  the  output  of  total  nitrogen. 
Hence,  the  percentage  steadily  decreases. 

No  explanation  can  be  given  for  the  irregular  results  obtained 
with  rabbit  1.  As  will  be  seen  in  the  tabulated  data  (Table  II), 
ammonia  nitrogen  was  entirely  absent,  or  present  in  amounts  too 
small  to  be  determined,  on  the  19th  and  22d,  but  markedly  in- 
creased until  the  24th,  when  it  represented  2.3  per  cent  of  the  total 
nitrogen.  On  the  25th  and  26th  it  was  still  high,  though  the 
percentage  had  slightly  fallen.    This  rabbit  was  a  small  animal, 


220 


Creatine  and  Creatinine  Metabolism 


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222         Creatine  and  Creatinine  Metabolism 


scarcely  full-grown,  and  was  in  very  poor  nutritive  condition  at 
the  beginning  of  the  experiment.  Possibly  this  may  explain  the 
relatively  large  output  of  ammonia  nitrogen  in  the  last  stages  of 
inanition.  It  is  interesting  to  note  that  the  elimination  of  crea- 
tine nitrogen  was  greater  in  this  animal  than  that  observed  in 
any  other  experiment,  thus  emphasizing  the  importance  of  the 
nutritive  condition  for  the  nitrogenous  metabolism. 

The  fact  that  starvation  does  not  induce  an  absolute  or  per- 
centage increase  in  the  elimination  of  ammonia  nitrogen  in  rab- 
bits, is  quite  in  contrast  with  the  findings  of  Brugsch,1  Cathcart,2 
Grafe3  and  others  on  men.  It  would  seem,  therefore,  that  her- 
bivora  are  either  not  subject  to  acidosis  to  the  same  extent  that 
omnivora  and  carnivora  are,  or  that  they  utilize  bases  other  than 
ammonia  for  the  neutralization  of  the  acid  products.  In  this 
connection,  the  suggestion  of  Burridge4 — that  creatine  may  serve 
as  a  neutralizing  agent  for  lactic  acid — is  an  interesting  but 
unverified  assumption. 

In .  subsequent  experiments  upon  rabbits  the  ammonia  deter- 
minations were  omitted. 

The  influence  of  a  carbohydrate  diet  upon  the  creatine-creatinine 
excretion  in  rabbits  during  inanition. 

Diet.  Numerous  attempts  were  made  to  give  starving  rab- 
bits dextrose  and  sucrose  by  the  stomach  tube  in  solutions  of 
varying  strengths,  but  the  results  were  invariably  very  unsatis- 
factory. Even  when  small  doses  of  the  sugar  solutions  were 
introduced  into  the  stomach  at  intervals  of  several  hours,  diar- 
rhoea was  evoked,  with  resulting  contamination  of  the  urine.  A 
more  serious  difficulty,  however,  was  the  fact  that  frequently 
the  kidney  excretion  was  practically  stopped  after  giving  the  sugar 
for  two  or  three  days,  and  the  animals  died  with  symptoms  of  uremic 
poisoning.    Hildebrandt5  observed  that  large  doses  of  dextrose 

1  Brugsch:    Zeitschr.  f.  exp.  Path.  u.  Therap.,  i,  pp.  419-30,  1905. 

2 Cathcart:    Biochem.  Zeitschr.,  vi,  pp.  122-23,  1907. 

3  Grafe:    Zeitschr.  f.  physiol.  Chem.,  lxv,  pp.  21-52,  1910. 

4 Burridge:    Journ.  of  Physiol.,  xli,  pp.  303-04,  1910. 

5 Hildebrandt:    Zeitschr.  f.  physiol.  Chem.,  xxxv,  pp.  141-52,  1902. 


Lafayette  B.  Mendel  and  William  C.  Rose  223 


exert  a  toxic  action  in  rabbits  fed  upon  a  diet  of  oats.  He  attrib- 
uted the  toxicity  to  the  production  of  large  amounts  of  oxalic 
acid  through  the  incomplete  combustion  of  the  sugar,  and  found 
that  the  addition  of  calcium  carbonate  to  the  diet  neutralized 
the  oxalic  acid  and  prevented  the  appearance  of  abnormal  symp- 
toms. He  makes  no  mention,  however,  of  a  decreased  kidney 
excretion. 

A  typical  experiment  illustrative  of  this  inhibition  of  kidney 
function,  is  summarized  in  Table  VI.  The  sugar-feeding  was 
begun  on  December  13.  On  the  15th,  the  animal  had  severe 
diarrhoea  and  contaminated  most  of  the  urine.  On  the  16th,  the 
inability  to  excrete  urine  was  very  evident.  The  complete  excre- 
tion for  twenty-four  hours  was  10  cc,  containing  only  0.04  gram 
of  total  nitrogen.  On  the  17th,  only  4  cc.  of  urine  were  excreted. 
On  the  18th,  an  attempt  was  made  to  increase  the  urine  elimina- 
tion by  giving  100  cc.  of  water  by  the  stomach  tube,  but  only 
50  cc.  of  a  very  dilute  urine  were  obtained,  containing  11  mgms. 
of  nitrogen  as  total  creatinine.  The  50  per  cent  retention  of  the 
water  could  not  have  been  due  to  a  depletion  of  the  tissue  mois- 
ture, for  the  animal  had  received  water  daily  throughout  the  exper- 
iment. Retention  is  further  indicated  by  the  fact  that  the  ani- 
mal began  to  increase  in  weight  on  the  15th,  and  continued  to 
increase  until  death  on  the  19th.  Before  death  severe  diarrhoea 
occurred,  accompanied  by  a  twitching  of  the  neck  and  shoulder 
muscles,  and  followed  by  coma. 

No  explanation  can  at  present  be  given  of  these  observations. 
Out  of  some  eight  or  ten  experiments  in  which  sugar  was  adminis- 
tered, satisfactory  results  were  obtained  only  once  (rabbit  6, 
Table  VII),  and  this  animal  differed  from  the  others  in  readily 
eating  loaf-sugar,  thus  obviating  the  necessity  of  giving  sugar 
solutions  by  the  stomach  tube. 

In  consequence  of  the  difficulties  associated  with  the  sugar- 
feeding,  this  diet  was  abandoned.  In  the  remaining  experiments 
(Tables  VIII  to  XI),  soluble-starch  suspended  in  water  was  given 
by  the  stomach  tube  with  very  satisfactory  results.  In  no  case 
did  this  diet  interfere  with  kidney  function. 

The  Composition  of  the  Urine.  In  the  following  experiments, 
as  in  those  previously  described,  creatine  is  a  constant  constit- 
uent of  the  urine  of  starving  rabbits.    In  rabbit  6,  creatine  did 


TABLE  VI. 


Rabbit  5 — Starvation;  carbohydrate  feeding. 


DATE 

BODY 
WEIGHT 

URINE 

DIET,  NOTES,  ETC. 

Vol- 
ume 

Specific  Reaction 
gravity  1  to  litmus 

Total 
N 

Creat- 
inine 
N 

Crea- 
tine 
N 

Nov. 

gms. 

cc. 

gms. 

mgms. 

mgms. 

30 

2240 

220 

1 

012 

Alkaline 

0.67 

30 

0 

Animal  ate  300  gms. 

carrots  and 50  gms. 

Dec. 

cracked  corn. 

1 

2260 

210 

1 

014 

Alkaline 

0.47 

27 

0 

Animal  ate  300  gms. 

carrots. 

2 

2240 

55 

1 

020 

Alkaline 

0.51 

32 

2 

No  food. 

3 

2160 

46 

1 

028 

Acid 

0.86 

28 

13 

No  food. 

4 

2080 

37 

1 

032 

Acid 

0.80 

28 

4 

No  food. 

5 

2040 

38 

1 

033 

Acid 

0.77 

29 

3 

No  food. 

6 

1980 

36 

1 

035 

Acid 

0.77 

27 

9 

No  food. 

7 

1920 

38 

1 

032 

Acid 

0.77 

30 

0 

No  food. 

8 

1860 

44 

1 

025 

Acid 

0.85 

27 

1 

No  food. 

9 

1800 

48 

1 

022 

Acid 

0.92 

23 

2 

No  food. 

10 

1740 

55 

1 

018 

Acid 

0.86 

22 

7 

No  food. 

11 

1700 

72 

1 

018 

Acid 

0.96 

23 

19 

No  food. 

12 

1640 

72 

1 

020 

Acid 

1.29 

23 

29 

No  food. 

13 

1580 

46 

1 

025 

Acid 

0.97 

24 

35 

Animal  was  given  15 

gms.  sucrose,  (loaf 

sugar) . 

14 

1510 

21 

Acid 

0.26 

22 

16 

30  gms.  sucrose  by 

stomach  -  sound  in 

50  per  cent  sol.  in 

six  equal  doses. 

15 

1550 

2* 

Acid 

0.02* 

3* 

0* 

35  gms.  ditto  in  seven 

equal  doses.  Part  of 

a  day's  urine.  Ani- 

mal had  diarrhoea. 

16 

1600 

10 

Acid 

0.04 

3 

1 

25  gms.  ditto  in  five 

equal  doses.  Com- 

plete kidney  excre- 

tion for  twenty-four 

hours. 

17 

1620 

4 

Acid 

2 

trace 

4  gms.  olive  oil  given 

by  stomach-sound. 

Complete  kidney  ex- 

cretion for  twenty- 

four  hours. 

18 

1620 

50 

1 

.010 

Neutral 

7 

4 

No   food.    100  cc. 

water  given  by 

stomach-sound. 

19 

1660 

Animal  died  in  after- 

noon   with  severe 

diarrhoea. 

*Not  a  complete  day's  urine,  see  notes. 


Lafayette  B.  Mendel  and  William  C.  Rose  225 


not  appear  in  appreciable  amounts  until  after  a  surprisingly  long 
period  of  inanition.  Small  amounts  were  periodically  excreted 
from  February  7  to  March  1,  but  the  quantities  were  too  small 
to  be  of  any  significance.  For  ten  days,  beginning  with  February 
14th,  the  animal  was  given  loaf-sugar  each  day,  but  the  only 
effect  observed  was  a  decrease  in  the  total  nitrogen  elimination, 
with  a  corresponding  increase  in  the  percentage  of  creatinine  nitro- 
gen. On  February  25,  starvation  was  resumed,  and  creatine  first 
appeared  in  significant  amount  on  March  2. 

This  remarkably  long  period  of  starvation  was  undoubtedly 
made  possible  by  the  excellent  nutritive  condition  of  the  animal, 
it  having  been  fed  liberally  for  several  weeks  before  beginning  the 
experiment.  The  store  of  glycogen  probably  prevented  the  ap- 
pearance of  much  urinary  creatine,  until  the  liver  and  muscles 
had  had  their  glycogen  supply  nearly  depleted. 

On  March  2,  sugar-feeding  was  again  begun  with  the  result 
that  the  creatine  nitrogen  fell  from  14  mgms.  on  the  2d,  to  zero 
on  the  5th.  After  two  days  of  fasting  the  creatine  nitrogen  again 
increased  until  it  was  more  than  twice  as  large  in  amount  as  the 
creatinine  nitrogen.  On  resuming  the  sugar  diet,  however,  it 
was  rapidly  reduced  to  zero.  On  March  13,  an  attempt  was  made 
to  give  a  protein  diet  consisting  of  coagulated  egg-white,  but  diar- 
rhoea resulted,  followed  by  the  death  of  the  animal  on  the  next 
day. 

Results  similar  to  these  were  obtained  on  rabbits  7,  8  and  9, 
each  of  which  received  the  soluble-starch  diet  instead  of  the  sugar. 
The  creatine  elimination  was  reduced  at  will  by  giving  an  abso- 
lutely nitrogen-  and  fat-free  carbohydrate  diet.  Usually  the  amount 
of  total  nitrogen  and  creatine  nitrogen  increased  the  first  day  of 
the  carbohydrate  administration,  but  rapidly  decreased  on  con- 
tinuing the  feeding.  Total  nitrogen  and  total  creatinine  appear 
to  have  a  common  source,  for  an  increase  or  decrease  in  the  latter  is 
always  accompanied  by  a  similar  change  in  the  former.  The  sig- 
nificance of  this  will  be  discussed  later. 

The  experiment  upon  rabbit  10  is  particularly  interesting 
because  of  the  results  following  the  administration  of  alcohol. 
The  protein-sparing  effect  of  small  doses  of  alcohol  is  well  known.1 

1For  the  literature  on  this  subject  cf.  Rosemann:  Oppenheimer' 's  Hand- 
buch  der  Biochemie,  iv,  part  1,  p.  433,  1909. 


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Lafayette  B.  Mendel  and  William  C.  Rose  231 


Recently,  Kochmann  and  Hall1  have  reported  the  results  of  exper- 
iments in  which  they  found  that  small  doses,  given  subcutaneously, 
greatly  prolonged  the  lives  of  starving  rabbits.  In  consideration 
of  these  results  it  seemed  possible  that  alcohol  might  exert  an  influ- 
ence on  the  creatine  excretion  similar  to  that  produced  by  carbo- 
hydrates. Hence,  in  the  experiment  upon  rabbit  10,  alcohol  was 
given  by  the  stomach-sound  for  two  days.  Rather  large  doses 
were  necessary  in  order  to  make  the  experiment  comparable  in 
fuel  value  with  those  in  which  carbohydrates  were  fed.  On  the 
17th  (Table  XI),  12  cc.  of  absolute  alcohol,  well  diluted  with  water, 
were  given  in  divided  dose  several  hours  apart.  The  animal  was 
rendered  intoxicated  for  two  or  three  hours  after  each  dose.  The 
urine  analysis  for  that  day  shows  that  the  total  nitrogen  was  in- 
creased, while  the  creatine  nitrogen  was  more  than  doubled. 
On  the  18th,  the  animal  was  kept  intoxicated  practically  all  day 
by  the  frequent  administration  of  alcohol,  with  the  result  that 
while  the  creatine  nitrogen  was  again  increased,  the  creatinine 
and  total  nitrogen  were  slightly  decreased.  Here  again  the  changes 
in  total  creatinine  elimination  are  associated  with  changes  in  the 
same  direction  in  total  nitrogen  elimination. 

That  the  increase  in  creatine  nitrogen  was  not  due  to  the  alco- 
hol per  se  seems  probable  from  the  observations  of  Mendel  and 
Hilditch  ('10),  who  found  no  increase  in  creatine  elimination  in 
dogs  even  after  prolonged  administration  of  alcohol.  The  increase 
must  therefore  have  been  due  solely  to  the  continued  lack  of 
carbohydrates.  At  the  same  time,  it  must  be  admitted  that 
the  doses  of  alcohol  given  to  the  rabbit  were  presumably  sufficient 
to  produce  the  so-called  toxic  nitrogenous  catabolism. 

On  the  19th,  administration  of  starch  suspensions  was  begun. 
Only  15  grams  of  soluble-starch  were  given  the  first  day,  which 
were  not  sufficient  to  prevent  the  creatine  and  total  nitrogen  from 
again  increasing.  On  the  20th  and  21st,  35  and  40  grams  respec- 
tively, of  starch  were  given,  with  the  result  that  the  creatine  elim- 
ination rapidly  disappeared,  accompanied  by  an  enormous 
decrease  in  total  nitrogen  excretion.  Starvation  and  starch- 
feeding  were  again  repeated  on  this  animal  with  results  similar 
to  those  previously  described. 

1  Kochmann  and  Hall:    Pfluger's  Archiv,  cxxvii,  pp.  280-356,  1909. 


232 


Creatine  and  Creatinine  Metabolism 


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Lafayette  B.  Mendel  and  William  C.  Rose  233 


From  the  foregoing  experiments,  it  can  be  definitely  stated 
that  a  carbohydrate  diet  absolutely  nitrogen-  and  fat-free,  produces 
a  marked  reduction  in  the  elimination  of  total  nitrogen  and  creatine 
in  starving  rabbits.  If  the  carbohydrate  feeding  is  continued  for 
two  or  three  days  creatine  disappears.  The  administration  of 
alcohol  produces  no  decrease  in  urinary  creatine  or  nitrogen. 

The  influence  of  fat  and  protein  upon  the  creatine-creatinine  excretion 
in  rabbits  during  inanition. 

Five  experiments  were  made  to  determine  the  effect  of  fat  and 
protein  feeding  upon  the  creatine-creatinine  elimination  in  starv- 
ing rabbits.  The  fat  diet  consisted  of  an  emulsion  of  73  per  cent 
peanut  oil,  2  per  cent  lecithin,  and  25  per  cent  water.1  Small 
doses  of  the  emulsion  were  given  at  intervals  of  several  hours  by 
the  stomach-sound.  The  proteins  used  were  casein  and  egg-white. 
The  results  of  the  experiments  will  be  seen  in  Tables  XII  to  XVI. 

In  rabbits  11,  12  and  13,  the  influence  of  the  fat  diet  without 
protein  was  investigated.  With  the  exception  of  animal  12,  abso- 
lutely no  reduction  in  creatine  or  total  nitrogen  followed  the  fat 
administration.  In  this  animal,  agar-agar  was  also  fed  with  the 
fat  emulsion  to  prevent  diarrhoea.  Hoffmann2  has  recently  shown 
that  agar-agar  can  be  decomposed  and  absorbed  by  rabbits,  but 
does  not  increase  the  sugar  in  the  urine  when  the  animals  are  phlor- 
hizinized.  He  believes  that  it  is  converted  into  fatty  acids  and 
absorbed  in  this  form.  There  is  no  reason  for  assuming  that  fatty- 
acids  arising  from  the  fermentation  of  agar  would  behave  differ- 
ently, in  regard  to  their  influence  on  the  creatine  elimination,  from 
those  liberated  in  fat  digestion.  Still  it  is  interesting  to  observe 
that  only  in  the  animal  that  received  agar  did  a  decrease  in  crea- 
tine and  nitrogen  elimination  follow  the  fat  feeding. 

It  is  impossible  to  state  the  degree  of  utilization  of  the  fat  diet. 
Diarrhoea  frequently  occurred  and  probably  resulted  in  poor 
absorption  on  these  days.  Herbivora  are,  furthermore,  suscept- 
ible to  lipuria  after  the  ingestion  of  large  amounts  of  fat.  In 
order  to  ascertain  definitely  the  amount  of  fat  actually  metabo- 

1  This  was  prepared  for  the  laboratory  by  Fairchild  Brothers  and  Foster, 
New  York. 

2 Hoffmann:    Inaug.  Diss.,  Halle,  pp.  27,  1910. 


234 


Creatine  and  Creatinine  Metabolism 


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Lafayette  B.  Mendel  and  William  C.  Rose  237 


lized,  it  would  therefore  be  necessary  to  determine  the  respiratory 
quotient.  It  is  certain,  however,  that  the  creatine  output  cannot 
be  reduced  in  rabbits  by  feeding  an  exclusive  fat  diet.  These  results 
are  in  accord  with  the  conclusions  of  Cathcart  ('09)  in  fasting  men. 

The  influence  of  a  mixed  protein  and  fat  diet  was  tested  in  rab- 
bits 14  and  15.  In  the  former,  from  the  12th  to  the  14th,  an  exclu- 
sively protein  diet  was  fed,  but  it  was  found  impractical  to  give 
sufficient  calories.  The  whites  of  eight  eggs  are  necessary  in  order 
to  yield  an  amount  of  energy  equivalent  to  40  grams  of  carbohy- 
drate. It  is  out  of  the  question  to  feed  so  large  an  amount  of 
protein  to  a  rabbit  without  producing  diarrhoea.  Hence,  from 
the  15th  to  the  18th,  sufficient  nitrogen  was  given  in  the  form  of 
protein  to  cover  the  nitrogenous  waste  of  the  animal,  and  the  calo- 
rific value  was  raised  by  the  addition  of  fat  emulsion.  No  reduc- 
tion in  the  creatine  elimination  occurred  on  this  diet  in  the  absence  of 
carbohydrates.  On  the  19th,  a  carbohydrate  diet  was  substituted 
for  the  protein-fat  diet,  with  the  result  that  the  outputs  of  crea- 
tine and  nitrogen  subsequently  decreased,  notwithstanding  the 
fact  that  the  animal  was  very  weak  and  emaciated.  Similar 
results  with  the  fat-protein  feeding  were  obtained  in  rabbit  15. 
On  account  of  diarrhoea  it  was  impossible  to  continue  the  carbo- 
hydrate diet  at  the  end  of  this  experiment  sufficiently  long  to  reduce 
the  creatine. 

The  results  obtained  after  protein  feeding  are  in  striking  con- 
trast to  those  briefly  reported  by  Osterberg  and  Wolf  ('08) .  These 
investigators  state  that  in  the  dog,  "the  creatine  produced  by 
starvation  is  inhibited  by  very  small  amounts  of  ingested  protein." 
Since  no  figures  are  given  in  the  preliminary  report,  it  is  not 
apparent  what  "very  small  amounts"  denote.  It  is  true  that  in 
rabbits  14  and  15  the  protein  ingested  was  calorifically  insufficient, 
but  the  amount  of  nitrogen  which  it  yielded  was  more  than  neces- 
sary to  compensate  the  nitrogenous  waste  of  the  animal.  That 
the  protein  was  absorbed  is  shown  by  the  greatly  increased  urin- 
ary nitrogen.  In  view  of  Osterberg  and  Wolf's  results,  a  decrease 
in  creatine  might  be  expected,  but  such  was  not  obtained.  In 
experiments  upon  fasting  men,  Cathcart  ('09)  found  that  the  addi- 
tion of  protein  produced  no  alteration  in  the  creatine  output  on  a 
fat  diet.  Since  this  is  true  for  herbivora  and  omnivora,  it  seems 
unlikely — in  the  absence  of  more  definite  data — that  carnivora 
differ  so  radically  in  their  metabolism. 


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240         Creatine  and  Creatinine  Metabolism 


The  want  of  available  carbohydrate  seems  to  be  the  primary 
factor  that  induces  the  appearance  of  urinary  creatine  in  starva- 
tion. It  is  interesting  in  this  connection  to  note  that  creatine  is 
also  excreted  in  many  conditions  where  carbohydrates  are  not 
properly  oxidized.  Reference  has  already  been  made  to  the  con- 
stant presence  of  creatine  in  the  urine  during  diabetes  mellitus. 
Recently  Krause  and  Cramer  ('10)  have  demonstrated  the  elim- 
ination of  creatine  in  dogs,  following  the  injection  of  phlorhizin. 
Similar  experiments  were  made  by  Cathcart  and  Taylor  ('10). 
The  latter  investigators  fed  their  animals  a  creatine-free  diet  and 
found  that  no  creatine  was  excreted  before  the  glycosuria,  pro- 
vided there  was  an  adequate  supply  of  carbohydrates.  If  the 
supply  was  very  small,  creatine  appeared  just  as  in  starvation. 
These  results  are,  of  course,  in  accord  with  the  findings  of  the  pres- 
ent investigations  on  fasting  rabbits. 

After  a  small  carbohydrate  intake,  Cathcart  and  Taylor  ('10) 
observed  an  output  of  creatine  as  the  result  of  the  injection  of 
phlorhizin.  The  creatine  was  present  only  so  long  as  the  glyco- 
suria persisted.  When,  however,  the  supply  of  ingested  carbohy- 
drates was  abundant,  in  spite  of  the  glycosuria,  creatine  was  not 
excreted.  After  substituting  fat  for  the  carbohydrate  of  the  diet, 
the  injection  of  phlorhizin  was  followed  by  a  much  less  intense 
glycosuria,  but  by  a  marked  excretion  of  creatine. 

It  would  appear  then  that  fat  in  the  food,  even  in  considerable 
amount,  does  not  prevent  protein  catabolism;  or  in  other  words 
cannot  replace  carbohydrates  under  the  experimental  conditions 
of  Cathcart  and  of  the  writers.  It  is  possible,  and  indeed  probable 
that  fat  may  replace  a  certain  amount  of  carbohydrate,  provided 
there  is  still  sufficient  carbohydrate  in  the  diet  to  exert  a  regulatory 
influence  over  certain  essential  metabolic  processes.  This  concep- 
tion is  in  accord  with  Rosenfeld's1  view  of  a  specific  action  of  car- 
bohydrates on  fat  metabolism.  This  author  believes  that  fats 
cannot  be  properly  burned  unless  carbohydrates  are  present. 
Similar  views  were  formulated  by  Landergren,2  who  found  that 
with  a  nitrogen  intake  of  approximately  one  gram,  the  output 

^osenfeld:  Verhandl.  d.  xxiv  Kongres.  f.  inner.  Med.,  Wiesbaden, 
pp.  279-83,  1907. 

2 Landergren:    Skand.  Arch.  f.  Physiol.,  xiv,  pp.  112-75,  1903. 


Lafayette  B.  Mendel  and  William  C.  Rose  241 


of  urinary  nitrogen  could  be  reduced  to  a  minimum  (3  to  4 
grams  per  day)  by  feeding  large  amounts  of  carbohydrates.  If, 
on  the  other  hand,  the  carbohydrates  of  the  diet  were  replaced 
by  fats  the  nitrogen  excretion  decreased  the  first  day,  but  increased 
on  the  second  and  third  days.  Landergren  believes  this  increase 
to  be  due,  not  to  a  specific  dynamic  action  of  the  fat,  but  to  a 
depletion  of  the  store  of  glycogen.  In  the  absence  of  carbohydrates 
the  fats  are  unable  to  exert  their  protein-sparing  effect.  It  may 
be  equally  true  that  creatine  cannot  be  properly  catabolized,  or 
converted  into  creatinine  in  the  absence  of  carbohydrate  food, 
or  that  the  cell  processes  themselves  are  radically  different  under 
such  conditions. 

So  we  might  expect  disturbances  of  the  liver  to  lead  to  the 
production  of  creatine  through  improper  glycogenic  function,  and 
in  fact  such  is  the  case.  Mellanby  ('07)  and  van  Hoogenhuyze 
and  Verploegh  ('08)  observed  the  excretion  of  creatine  in  hepatic 
diseases,  notably  carcinoma;  and  Underhill  and  Kleiner  ('08) 
found  an  increased  excretion  of  creatine  in  the  urines  of  dogs  poi- 
soned with  hydrazine — a  substance  which  is  known  to  have  a 
specific  action  upon  the  cytoplasm  of  the  parenchymatous  cells 
of  the  liver.  The  internal  administration  of  chloroform  which, 
as  Clark1  has  shown,  produces  great  degenerative  changes  in  the 
hepatic  cells,  induces  the  appearance  of  creatine  in  the  urine  (cf. 
Howland  and  Richards,  '09,  and  Lindsay,  '11).  More  recently 
van  Hoogenhuyze  and  ten  Doeschate  ('11)  have  reported  the  pres- 
ence of  large  amounts  of  creatine  in  eclampsia.  Likewise,  London 
and  Boljarski  ('09),  Foster  and  Fisher  ('11),  and  others,  found 
creatine  in  the  urine  of  Eck  fistula  dogs,  where  the  hepatic  func- 
tions are  entirely  removed. 

It  is  true  that  the  presence  of  creatine  in  these  conditions  may 
be  explained  by  assuming  that  the  liver  is  the  organ  that  brings 
about  the  conversion  of  creatine  into  creatinine,  and  that  when 
the  hepatic  cells  are  diseased  or  removed  from  the  sphere  of  action 
by  artificial  alterations  of  the  circulation,  this  conversion  cannot 
occur.  But  if  this  assumption  were  correct,  creatine  alone  should 
be  excreted  in  animals  with  Eck  fistulas,  and  creatinine  should 
be  entirely  absent — a  condition  which  has  never  been  attained. 

1  Clark:  Proc.  Roy.  Soc.  Edinb.,  xxix,  pp.  418-26,  1909. 


242 


Creatine  and  Creatinine  Metabolism 


Van  Hoogenhuyze  and  ten  Doeschate  ('11)  attempt  to  explain 
the  presence  of  creatine  in  eclampsia  on  the  ground  that  the  liver 
is  diseased,  and  is,  therefore,  unable  to  bring  about  the  dehydra- 
tion. As  evidence  of  the  liver  disorder  they  refer  to  the  post 
mortem  findings  of  capillary  hemorrhages  in  the  liver  parenchyma, 
and  to  the  greatly  reduced  tolerance  for  sugar  in  such  patients. 
May  not  the  reduced  tolerance,  and  hence  the  lowered  glycogenic 
function,  be  the  factor  occasioning  the  excretion  of  creatine, 
rather  than  the  condition  of  the  liver  per  se?  The  presence  of 
creatine  in  the  urine  during  pregnancy  as  noted  by  Murlin  ('08-09), 
Longridge,1  and  others,  can  readily  be  explained  on  this  assump- 
tion, for  Bar2  found  a  greatly  reduced  tolerance  for  sugar  in  preg- 
nant women.  Certainly,  from  a  consideration  of  the  data  which 
have  been  collected  in  regard  to  the  effect  of  carbohydrates  on 
creatine  elimination,  it  is  just  as  probable  that  the  real  factor  in 
the  production  of  creatine  in  hepatic  disease  is  a  disturbance  of 
carbohydrate  metabolism. 

To  further  test  the  influence  of  the  amount  of  glycogen  and  of 
the  condition  of  the  hepatic  cells  upon  creatine  excretion,  two  exper- 
iments were  undertaken  upon  dogs.  In  the  first,  an  attempt  was 
made  to  remove  the  glycogen  by  repeated  injections  of  phlorhizin 
in  a  starving  animal.  It  was  hoped  that  after  repeated  phlorhizin 
administration  the  glycogen  could  be  largely  removed  from  the 
animal,  with  the  result  that  creatine  would  continue  to  be  present 
in  the  urine  after  the  glycosuria  had  disappeared.  In  the  second 
experiment  a  fasting  animal  was  given  frequent  injections  of  phos- 
phorus oil,  in  order  to  cause  degeneration  of  the  hepatic  cells. 
The  protocols  are  summarized  in  Tables  XVII  and  XVIII. 

The  phlorhizinized  animal  received  the  drug  subcutaneous! y  in 
sodium  carbonate  solution,  according  to  the  procedure  recom- 
mended by  Lusk.3  Three  doses  of  2  grams  each  were  given  on 
January  20  and  21,  and  one  dose  of  2  grams  on  the  22d.  Sugar 
continued  to  be  excreted  until  January  27,  whereupon  the  crea- 
tine estimations  were  begun  after  the  glycosuria  ceased.  As  will 
be  seen,  creatine  alternately  appeared  and  disappeared.  On 

longridge:    Cited  by  van  Hoogenhuyze  and  ten  Doeschate  ('11). 

2  Bar  (cited  by  Lequex):  L'Obstetrique,  iii,  p,  506,  1910. 

3  Lusk:    Amer.  Journ.  of  Physiol.,  xxii,  pp.  164-65,  1908. 


Lafayette  B.  Mendel  and  William  C.  Rose 


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244  Creatine  and  Creatinine  Metabolism 


February  1,  the  amount  of  creatine  nitrogen  exceeded  the  creatinine 
nitrogen.  Two  days  later  creatine  was  entirely  absent.  On 
February  10,  it  was  again  excreted  and  continued  to  be  present 
in  increasing  amount  until  February  12,  when  it  exceeded  the 
amount  of  preformed  creatinine  by  over  200  per  cent.  Unfor- 
tunately, the  urines  of  the  next  two  days  were  contaminated  with 
feces,  and  analyses  were  not  made. 

No  adequate  explanation  of  this  irregular  excretion  of  creatine 
is  apparent.  It  is  probable  that  the  amount  present  was  due  to 
the  fasting,  though  the  injection  of  phlorhizin  may  have  been  a 
contributing  factor  in  the  partial  removal  of  glycogen.  In  regard 
to  the  disappearance  and  subsequent  reappearance  of  creatine, 
the  observation  of  Pfliiger  and  Junkersdorf1 — that  a  re-formation 
of  glycogen  from  protein  occurs  in  phlorhizinized  dogs — is  of  par- 
ticular interest.  If  we  accept  this  as  an  established  fact,  the 
sequence  of  changes  in  creatine  elimination  can  be  readily  under- 
stood. The  phlorhizin  and  starvation  removed  sufficient  glycogen 
to  leave  the  animal  in  need  of  carbohydrate,  and  creatine  imme- 
diately appeared  in  significant  amount.  A  re-formation  of  gly- 
cogen caused  the  subsequent  disappearance  of  creatine,  which 
did  not  again  appear  until  the  glycogen  supply  had  again  been 
depleted  on  February  10.  Of  course  this  explanation  is  entirely 
theoretical  and  tentative.  It  is  well  known  that  dogs  are  especially 
hard  to  render  glycogen-free.  They  seem  to  retain  or  replenish 
at  least  part  of  their  store,  in  spite  of  varied  experimental  attempts 
to  exhaust  it.  This  fact  may  explain  their  ability  to  endure  fast- 
ing usually  for  remarkably  long  periods  without  serious  conse- 
quences,2 and  without  the  excretion  of  large  amounts  of  creatine 
or  nitrogen. 

In  the  experiment  on  the  phosphorus-poisoned  dog  (Table 
XVIII),  creatine  was  constantly  present  in  the  urine  in  significant 
amounts.  Beginning  with  May  28  and  continuing  until  the  death 
of  the  animal,  the  amount  of  nitrogen  excreted  in  this  form  greatly 
exceeded  that  as  creatinine.    This  appearance  of  creatine  in  con- 

1  Pfliiger  and  Junkersdorf:    Pfliiger's  Archiv,  cxxxi,  pp.  201-301,  1909. 

2  Howe,  Mattill  and  Hawk  (this  Journal,  vii,  p.  xlvii,  1910)  have  recorded 
the  longest  fast  on  record,  i.e.,  117  days.  On  the  117th  day  the  dog  showed 
a  loss  of  63  per  cent  of  body  weight.  The  animal  was  carefully  fed  and 
brought  back  to  nitrogen  equilibrium. 


Lafayette  B.  Mendel  and  William  C.  Rose  245 


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246         Creatine  and  Creatinine  Metabolism 

siderable  amounts  is  of  particular  interest  in  view  of  the  recently 
published  paper  of  Frank  and  Isaac.1  These  investigators  found 
that  during  phosphorus  poisoning,  the  amount  of  sugar  in  the 
blood  practically  disappears.  If  carbohydrates  are  necessary 
for  normal  cellular  metabolism,  or  for  the  conversion  of  creatine 
into  creatinine,  as  has  been  suggested,  then  creatine  would  be 
expected  under  such  conditions.  Lusk  ('07)  was  unable  to  detect 
any  significant  change  in  creatinine  output  after  phosphorus 
poisoning,  in  one  experiment  on  a  fasting  dog.  No  creatine 
estimations  were  reported.  Lefmann  ('08)  concluded  that  the 
creatinine  elimination  was  increased  during  the  poisoning  not 
accompanied  by  fasting,  while  the  creatine  output  remained  unal- 
tered. His  data,  however,  are  so  irregular  that  definite  conclu- 
sions are  impossible. 

The  parallelism  between  total  creatinine  and  total  nitrogen  elim- 
ination is  very  striking  in  the  phosphorus-intoxicated  dog  as  well 
as  in  the  experiments  previously  described.  An  increase  in  total 
creatinine  is,  without  exception,  accompanied  by  an  increase  in 
total  nitrogen.  There  is,  however,  one  difference  in  the  results 
obtained  on  dogs  as  contrasted  with  those  obtained  on  rabbits. 
In  dogs  the  excretion  of  creatine  is  usually  accompanied  by  a 
decrease  in  creatinine,  though  not  commensurate  with  the  increase 
in  creatine;  while  in  rabbits  the  creatinine  is  comparatively  con- 
stant in  amount,  or  shows  only  a  very  slight  tendency  to  decrease 
during  the  last  days  of  the  fasting  period.  The  behavior  of  dogs 
would  seem  to  indicate  that  creatine  and  creatinine  have  the  same 
origin  in  the  organism,  and  that  while  the  production  of  creatine 
increases  as  starvation  progresses,  less  and  less  conversion  to  the 
anhydride  is  accomplished. 

The  protocols  of  Cathcart  ('09)  show  a  decrease  in  creatinine 
excretion  in  man,  coincident  with  the  increase  in  creatine,  but 
contrary  to  the  present  findings  no  parallelism  exists  between  the 
elimination  of  total  creatinine  and  total  nitrogen  in  his  experiments. 
Moreover,  he  makes  no  reference  to  the  possible  importance  of 
carbohydrates  in  bringing  about  the  conversion  of  creatine  to  cre- 
atinine. 

1  Frank  and  Isaac:    Arch.  f.  exp.  Path.  u.  Pharm.,  lxiv, pp.  274-92,  1911. 


Lafayette  B.  Mendel  and  William  C.  Rose  247 


GENERAL  DISCUSSION. 

Two  fundamental  facts  are  emphasized  by  the  experiments 
recorded:  (1)  An  increase  in  the  elimination  of  total  creatinine 
(i.e.,  creatine  plus  creatinine)  is  always  accompanied  by  an  increase 
in  the  output  of  total  nitrogen;  and  (2)  Carbohydrates,  in  contrast  to 
the  other  food-stuffs,  are  capable  of  preventing  the  excretion  of  crea- 
tine, and  are  therefore  indispensable  for  normal  creatine-creatinine 
metabolism. 

Although  an  increased  output  of  nitrogen  in  the  form  of 
creatine  and  creatinine  is  associated  with  a  rise  in  total  nitrogen 
elimination,  it  by  no  means  follows  that  the  reverse  is  true. 
It  is  necessary  to  remember  that  under  ordinary  conditions  we 
have  in  the  animal  body  three  sources  of  urinary  nitrogen, 
namely:  food  nitrogen,  reserve  nitrogen,  and  tissue  nitrogen. 
During  fasting  the  food  nitrogen  does  not  enter  into  the  problem. 
The  urinary  nitrogen  in  inanition,  therefore,  has  its  origin  in  (a), 
reserve  nitrogen  (variously  termed  circulating  protein,  Vorratsei- 
weiss,  Reserveeiweiss,  Zelleneinschluss,  labiles  Eiweiss,  by  differ- 
ent writers);  and  (6)  disintegration  of  organized  body  tissue — 
so-called  endogenous  nitrogenous  metabolism.  If  the  former 
were  its  source,  no  accompanying  increase  in  total  creatinine  should 
occur,  for  it  is  probable  that  this  form  of  nitrogen  is  metabolized 
just  as  the  (exogenous)  food  nitrogen.  When,  on  the  other  hand, 
it  becomes  necessary  for  the  tissues  to  disintegrate  to  furnish 
energy  for  the  organism,  an  increase  in  creatine  or  creatinine  neces- 
sarily occurs.  The  most  abundant  tissue,  and  one  which  suffers 
great  loss  in  weight,  namely,  muscle  tissue,  contains  considerable 
creatine.  This  will  be  liberated  and  might  be  expected  to  appear 
in  the  urine  either  unaltered  or  as  creatinine. 

But  this  is  not  the  only  factor  involved.  Considerable  evidence 
has  been  accumulated  in  recent  years,  indicating  that  creatine  is 
a  product  of  endogenous  metabolism,  and  that  an  increased  for- 
mation of  creatine  occurs  when  the  tissue  catabolic  processes 
are  accelerated.  There  is  evidence,  also,  that  creatine  can  in  part 
disappear  in  the  body,  and  in  part  be  converted  into  creatinine. 
Of  importance  in  this  connection,  is  the  work  of  Gottlieb  and  his 
collaborators.  Gottlieb  and  Stangassinger  ('07,  '08),  and  Stan- 
gassinger  ('08),  found  that  during  the  autolysis  of  muscle  and  other 


248         Creatine  and  Creatinine  Metabolism 


organs,  a  formation  of  creatine  occurs.  The  creatine  so  arising, 
as  well  as  creatine  added  to  the  autolytic  mixture  is,  by  the  action 
of  an  enzyme,  partially  converted  into  creatinine.  Both  creatine 
and  creatinine  are  by  long  continued  autolysis,  destroyed  through 
the  action  of  specific  enzymes  (creatase  and  creatinase).  The  very 
complex  curves  representing  the  amounts  of  creatine  and  creatinine 
in  an  autolytic  mixture  depend,  therefore,  upon  the  balance  between 
formation,  conversion,  and  destruction  of  these  substances. 

The  results  of  Gottlieb's  experiments  have  been  severely  criti- 
cised by  Mellanby  ('08),  who  concluded  that  when  the  autolytic 
experiments  were  kept  rigorously  free  from  bacteria,  and  when 
precautions  were  taken  to  prevent  the  conversion  of  creatine  into 
creatinine  by  heating,  no  change  occurred  during  autolysis.  The 
experiments  have,  however,  been  repeated  with  improved  technic 
by  Rothmann  ('08)  and  van  Hoogenhuyze  and  Verploegh  ('08), 
and  results  obtained  similar  to  those  of  Gottlieb. 

Whether  one  accepts  Gottlieb's  or  Mellanby's  experiments,  the 
fact  remains  that  in  muscle  tissue  an  increased  production  of  crea- 
tine may  actually  occur  under  appropriate  conditions.  Weber 
('07)  and  Howell  and  Duke  ('08)  found  that  the  beating  heart 
liberates  creatine  into  the  perfusion  fluid.  Weber  also  observed 
that  the  creatinine  excretion  is  considerably  higher  in  dogs  poi- 
soned with  cinchonine,  indicating  that  increased  tonus  may  lead 
to  creatine  or  creatinine  formation.  Likewise  Graham-Brown 
and  Cathcart  ('08)  found  that  stimulation  of  isolated  frog  muscles 
brought  about  an  increase  of  from  7  to  13  per  cent  in  the  crea- 
tine content.  Van  Hoogenhuyze  and  Verploegh  ('08)  found  the 
excretion  of  creatinine  to  be  greater  during  the  day,  when  the  mus- 
cle wear  and  tear  is  increased,  than  during  the  night  when  the  mus- 
cle tonus  is  reduced. 

More  recently  Pekelharing  and  van  Hoogenhuyze  ('10a)  have 
demonstrated  an  increased  formation  of  creatine  in  the  muscle 
during  rigor  caloris,  rigor  mortis,  and  heightened  tonus.  These 
authors  believe  that  during  tonus  muscle  protein  is  decomposed 
to  furnish  energy,  and  that  creatine  is  a  product  of  this  endogenous 
catabolism.  The  sources  of  the  energy  utilized  for  the  maintenance 
of  tonus  and  for  the  performance  of  ordinary  muscular  work  are, 
according  to  this  view,  entirely  different.  If  this  theory  is  correct, 
an  increased  formation  of  creatine  should  occur  during  fasting 


Lafayette  B.  Mendel  and  William  C.  Rose  249 


after  the  supply  of  non-nitrogenous  energy-yielding  food-stuffs 
and  of  nitrogenous  reserve  material  has  been  depleted,  and  should 
not  occur  until  such  a  depletion  has  been  effected.  The  theory 
and  the  facts  obtained  in  the  experiments  already  described  are, 
therefore,  entirely  in  accord. 

The  numerous  other  observations  of  creatine  excretion  in  condi- 
tions associated  with  wasting  of  muscle  tissue,  as  in  fevers  (Shaffer, 
'08b;  and  van  Hoogenhuyze  and  Verploegh,  '08),  during  the  post 
partum  resolution  of  the  uterus  (Shaffer,  '08b;  and  Murlin,  '08-'09), 
and  in  muscular  disease  (Levene  and  Kristeller,  '09),  all  point  to 
the  same  conclusion,  namely,  that  whenever  muscle  protein  is 
decomposed,  creatine  is  a  product  of  the  disintegration.  Further 
evidence  of  this  will  be  found  in  a  marked  increase  of  the  creatine 
content  of  muscle  during  starvation,  in  hens  and  rabbits.1 

At  the  present  time  the  most  probable  explanation  of  the  pro- 
duction of  creatine  and  creatinine  may  be  sought  in  the  catabolism 
of  the  tissues  (i.e.,  endogenous  metabolism).  Under  appropriate 
nutritive  conditions,  the  small  amount  of  creatine  arising  from 
muscle  wear  and  tear,  is  converted  into  creatinine  and  excreted. 
When,  however,  an  undue  creatine  production  occurs,  the  conver- 
sion to  creatinine  may  become  inadequate,  and  creatine  as  such 
appear  in  the  urine.  Possibly  some  may  be  oxidized  and  not 
appear  at  all.  In  this  case  creatine  and  creatinine  would  be  anal- 
ogous to  uric  acid  and  represent  a  balance  between  formation  and 
destruction.  They  would  then  be  intermediary  rather  than 
end  products.  These  views  are  represented  in  the  accompanying 
scheme. 


Tissue  N 


Food  N 


Reserve  N 


Creatine 


No  Creatine 


No  Creatine 


(Oxidized) 
? 


Urinary 
Creatinine 


Urinary 
Creatine 


irrhe  experimental  proof  of  this  will  be  furnished  in  the  next  paper. 


250         Creatine  and  Creatinine  Metabolism 

It  may  be  objected  that  if  this  theory  were  true  creatine  intro- 
duced per  os  or  parenterally  should  ordinarily  be  converted  into 
creatinine,  and  that  this  is  contrary  to  the  observations  of  most 
investigators  (Folin,  '06,  Klercker,  '06  and  '07,  Lefmann,  '08). 
But  this  does  not  follow  any  more  than  that  creatine  arising  dur- 
ing starvation  should  be  converted  into  creatinine.  It  is  possible 
that  the  organism  is  capable  of  converting  only  a  definite  amount 
of  creatine  into  the  anhydride,  and  that  when  this  amount  is 
exceeded,  unaltered  creatine  appears  in  the  urine.  On  the  other 
hand,  a  slight  conversion  was  observed  by  van  Hoogenhuyze  and 
Verploegh  ('08).  In  the  most  recent  paper  along  this  line,  Pekel- 
haring  and  van  Hoogenhuyze  ('10b)  found  that  creatine  parenter- 
ally introduced  into  rabbits  and  dogs,  was  partly  transformed  into 
creatinine,  partly  oxidized,  and  partly  excreted  unaltered.  It 
must  be  remembered  also  that  in  injection  experiments  the  cir- 
culation may  be  so  flooded  with  creatine,  that  there  is  not  suffi- 
cient time  for  conversion  before  elimination  occurs.  In  many 
of  the  earlier  experiments,  where  creatine  was  given  by  mouth, 
it  is  not  improbable  that  it  was  largely  decomposed  by  bacteria 
in  the  alimentary  canal  (cf.  Czernecki,  '05,  and  Nawiasky,  '08). 
Plimmer,  Dick  and  Lieb  ('09-' 10),  in  experiments  in  man,  found 
that  2.5  grams  of  creatine  had  to  be  given  by  mouth  before  any 
could  be  recovered  in  the  urine.  This  may  serve  to  explain  the 
entire  disappearance  of  creatine  in  many  feeding  experiments, 
and  its  failure  to  increase  the  creatinine  output. 

With  our  present  knowledge,  it  is  impossible  to  formulate  any- 
thing definite  as  to  the  chemical  processes  by  which  creatine 
arises  in  tissue  catabolism.  The  striking  similarity  between  its 
structure  and  the  structures  of  many  other  substances  occurring 
in  muscle  tissue,  or  derived  from  proteins,  indicates  that  its  origin 
in  tissue  catabolism  is  by  no  means  inconceivable.  Attempts, 
however,  to  associate  these  compounds  with  creatine  and  creatinine 
experimentally,  have  thus  far  been  unsuccessful  or  doubtful  (cf. 
Burian,  '05,  Jaffe,  '06,  Achelis,  '06,  Dorner,  '07,  and  Lefmann,  '08). 

It  is  difficult  to  form  any  chemical  picture  of  the  influence  car- 
bohydrates may  have  in  preventing  the  excretion  of  creatine.  As 
already  suggested,  they  may  be  necessary  for  the  conversion  of 
creatine  into  creatinine,  or  in  their  presence  creatine  may  be  more 
readily  oxidized  and  excreted  as  urea.    Again,  the  tissue  cells 


Lafayette  B.  Mendel  and  William  C.  Rose  251 


may  not  functionate  properly  when  the  normal  amount  of  carbo- 
hydrate food  is  wanting,  and  in  this  case  the  elimination  of  crea- 
tine would  be  analogous  to  the  production  of  the  acetone  bodies, 
which  is  also  inhibited  by  the  administration  of  carbohydrates. 
More  work  will  be  necessary  to  elucidate  these  problems.  With- 
out question  the  metabolism  of  creatine  is  intimately  associated  with 
carbohydrate  metabolism. 

SUMMARY. 

1.  The  excretion  of  creatine  induced  by  starvation,  is  inhib- 
ited in  rabbits  by  feeding  a  diet  of  carbohydrates  absolutely  free 
from  proteins  and  fats.  When  the  carbohydrates  are  given  in 
liberal  amounts,  creatine  entirely  disappears  from  the  urine. 

2.  The  creatine  elimination  is  not  reduced  by  feeding  a  diet 
of  fat  alone,  or  by  a  diet  of  fat  and  protein. 

3.  Experimental  interference  with  carbohydrate  metabolism 
leads  to  the  elimination  of  creatine.  After  phlorhizin  diabetes 
which  depletes  the  store  of  carbohydrates,  and  during  phosphorus 
poisoning,  which  disturbs  the  glycogenic  functions,  the  output  of 
creatine  in  dogs  is  decidedly  increased. 

4.  An  increase  in  the  output  of  creatine  plus  creatinine  (total 
creatinine),  is  always  accompanied  by  an  increase  in  total  nitrogen 
elimination.  This  parallelism  of  total  creatinine  and  total  nitro- 
gen outputs  in  inanition  and  with  nitrogen-free  diets  is  ascribed 
to  a  common  source,  namely,  true  tissue  or  endogenous  metabolism. 
The  metabolism  of  exogenous  or  reserve  proteins  is  not  accom- 
panied by  the  production  of  creatine  or  creatinine. 

5.  The  intimate  relation  of  creatine  excretion  (or  the  failure 
of  conversion  into  creatinine)  to  carbohydrate  metabolism,  is 
discussed  in  detail. 

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Reprinted  from  The  Journal  of  Bioloqical  Chemistry,  Vor,.  X,  No.  3,  1911 


EXPERIMENTAL  STUDIES  ON  CREATINE  AND 
CREATININE. 

n.    INANITION  AND  THE  CREATINE  CONTENT  OF  MUSCLE.* 

By  LAFAYETTE  B.  MENDEL  and  WILLIAM  C.  ROSE. 

(From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  August  14,  1911.) 

Comparatively  few  investigations  have  been  made  in  regard 
to  the  creatine  content  of  muscle  in  normal  and  abnormal  condi- 
tions. Such  data  as  we  have  were,  for  the  most  part,  obtained 
with  the  older  and  inadequate  analytical  method  of  Neubauer  and 
are  consequently  usually  unreliable.  Many  of  these  researches 
were  undertaken  with  a  view  of  determining  the  influence  of 
muscular  work  on  the  content  of  creatine,  but  because  of  the 
errors  in  technic  the  results  have  been  widely  different. 

Liebig  ('47)  was  the  first  to  begin  systematic  studies  on  the  effect 
of  activity  on  tissue  composition.  In  1847  he  made  his  classi- 
cal investigation  of  the  creatine  content  of  muscle  in  fatigue 
and  found  that  the  muscle  of  a  fox  killed  in  the  chase  contained 
ten  times  the  amount  of  creatine  present  in  an  equal  weight  of 
muscle  from  a  resting  animal.  Sarokow  ('63)  also  found  an  in- 
crease of  creatine  after  work.  He  believed  that  coincident  with 
the  formation  of  creatine,  the  muscular  activity  brought  about  a 
partial  conversion  into  creatinine.  A  similar  increase  in  creatine 
was  reported  by  Sczelkow  ('66)  as  the  result  of  tetanus,  while 
rest  was  supposed  to  produce  a  decrease.  On  the  other  hand  Naw- 
rocki  ('66)  in  experiments  on  dogs,  found  no  difference  in  the  crea- 
tine content  of  tetanized  and  resting  muscle. 

The  results  of  Voit  ('68)  were  again  different.  In  experiments 
on  frogs,  he  decided  that  tetanus  produced  a  decrease  in  the 

1  The  experimental  data  are  taken  from  the  thesis  presented  by  William 
C.  Rose  for  the  degree  of  Doctor  of  Philosophy,  Yale  University,  1911. 


255 


256         Creatine  and  Creatinine  Metabolism 


creatine  content  of  muscle.  According  to  this  investigator  the 
creatine  is  transformed  during  work  to  a  substance  not  identical 
with  creatinine. 

The  most  consistent  of  the  earlier  results  on  the  effect  of  work 
were  obtained  by  Monari  ('87)  who  found  that  an  increase  in 
creatine  invariably  occurred  in  fatigue,  and  that  coincident  with 
the  increase  a  much  larger  amount  of  creatinine  was  detectable. 
The  protocols  of  Monari  appear  very  conclusive,  and  were  accepted 
and  frequently  quoted  in  subsequent  papers.  It  seems  prob- 
able now  from  the  investigations  of  Mellanby  ('07-  08),  that  much 
of  the  precipitate  which  Monari  weighed  as  pure  creatine,  was 
composed  of  other  substances.  The  creatinine  obtained  by  him 
was  undoubtedly  derived  from  creatine  in  the  evaporation  of 
the  acid  tissue  extracts.  That  preformed  creatinine  does  not 
exist  in  freshly  killed  muscle  is  definitely  proven  by  the  researches 
of  Grindley  and  Woods  ('06-'07),  Mellanby  ('07-'08),  and  Paton 
C09-'10). 

Since  the  introduction  of  the  Folin  colorimetric  method  for  the 
estimation  of  creatinine,  several  papers  have  appeared  on  the  influ- 
ence of  activity  on  muscle  creatine.  Weber  ('07)  found  that 
work  caused  a  slight  decrease  in  the  creatine  content,  and  that 
the  beating  heart  gave  off  creatine  or  creatinine  into  the  perfusion 
fluid.  According  to  this  author,  less  creatine  was  present  in  degen- 
erated than  in  normal  muscle.  Mellanby  ('07-'08),  on  the  other 
hand,  found  that  the  performance  of  muscular  work,  as  well  as 
the  survival  of  isolated  muscle,  leaves  creatine  unaffected.  No 
change  in  the  creatine  content  was  found  by  von  Fiirth  and 
Schwarz  ('11)  after  tetanizing  the  hind-leg  muscles  of  a  dog  for 
more  than  an  hour. 

Graham-Brown  and  Cathcart  ('08)  report  that  stimulation  pro- 
duces an  increase  in  the  amount  of  total  creatinine  in  isolated 
frog  muscles,  whereas  it  leads  to  a  slight  decrease  when  the  circu- 
lation is  left  intact.  In  a  later  communication,  these  authors 
(Graham-Brown  and  Cathcart,  '09)  report  the  results  of  studies 
made  on  rabbits,  in  which  they  find  that  with  the  circulation  in- 
tact, stimulation  induces  a  constant  though  small  decrease  in 
the  amount  of  total  creatinine  (i.e.,  creatine  plus  creatinine). 

Practically  no  data  have  been  published  on  the  effect  of  factors 
other  than  activity  on  the  creatine  content  of  muscles.    By  the 


Lafayette  B.  Mendel  and  William  C.  Rose  257 


use  of  the  Neubauer  method  Demant  (79)  observed  an  increase 
in  the  percentage  of  creatine  in  the  breast  muscles  of  pigeons  dur- 
ing starvation.  All  figures  given  by  this  author  are  much  lower 
than  those  obtained  by  the  use  of  the  Folin  method.  From  the 
analysis  of  the  muscle  of  a  single  starving  rabbit  Dorner  ('07, 
p.  261),  using  the  Folin  method,  concluded  that  there  was  a  decrease 
in  creatine  during  inanition.  Recently,  in  a  preliminary  report, 
Howe  and  Hawk  ('11)  claim  that  a  very  marked  reduction  in  the 
creatine  of  dog's  muscles  is  brought  about  by  fasting. 

Such  studies  are  of  prime  importance  in  determining  the  origin 
of  urinary  creatine  and  creatinine.  If  it  is  universally  true  that 
a  decrease  in  the  creatine  content  of  muscle  occurs  during  inani- 
tion, the  creatine  found  in  the  urine  during  starvation  must  have 
its  origin  in  a  "  washing-out"  of  muscle  creatine.  If,  on  the 
other  hand,  the  accelerated  endogenous  metabolism  during  star- 
vation occasions  an  increased  formation  of  creatine,  as  has  been 
suggested  in  a  previous  paper  (cf.  Mendel  and  Rose  '11),  then 
urinary  creatine  and  creatinine  would  represent  local  metabolic 
end-products,  rather  than  substances  merely  washed  out  of  the 
tissues.  The  excess  of  creatine  produced  during  such  a  process 
might  be  entirely  excreted  or  oxidized,  or  partly  retained  in  the 
muscle.  The  muscle  analyses  would  then  show  either  no  change 
or  an  increase  in  the  creatine  content,  but  never  a  decrease. 

To  determine  this  question  a  series  of  analyses  was  made  of 
muscle  tissue  removed  from  normal  and  starving  rabbits  and  hens, 
the  results  of  which  are  summarized  in  Tables  II  and  III.  The 
animals  were  allowed  to  starve  for  varying  lengths  of  time  and 
killed  by  bleeding.  To  avoid  errors  which  might  arise  from  differ- 
ences in  the  amount  of  creatine  in  different  muscles,  similar  mus- 
cles were  always  selected  in  the  control  and  experimental  animals. 
In  the  rabbits,  the  muscle  tissue  from  the  hind  legs  and  back  was 
completely  removed,  freed  as  much  as  possible  from  connective 
tissue,  and  thoroughly  ground  in  a  hashing  machine.  From  the 
uniform  mixture  samples  were  rapidly  weighed  for  the  analyses. 
In  the  fowl,  only  the  pectoral  muscles  were  used. 

The  total  creatinine  was  estimated  according  to  the  procedure 
of  Mellanby.1 

lMellanby:   Journ.  of  Physiol.,  xxvi,  pp.  453-4,  1907-8. 

ft, 


258         Creatine  and  Creatinine  Metabolism 


For  this  purpose,  the  finely  ground  muscle  was  killed  by  covering  with 
95  per  cent  alcohol.  The  alcohol  was  poured  off  through  silk  gauze,  the 
muscle  pressed  out,  and  repeatedly  extracted  by  shaking  with  five  portions 
of  water,  about  half  an  hour  being  allowed  for  the  extraction  with  each  por- 
tion. The  alcoholic  and  watery  extracts  were  then  combined  and  evapor- 
ated to  dryness  on  the  water-bath.  The  residue  was  extracted  five  times 
with  75  per  cent  alcohol,  which  removed  the  creatine  and  creatinine,  but 
left  most  of  the  protein  behind.  The  alcohol  was  removed  by  evaporation, 
the  solution  made  up  to  a  known  volume  with  water — usually  15G  cc. — fil- 
tered, and  the  total  creatinine  determined  on  10  cc.  portions  by  the  Folin- 
Benedict-Myers  method.  Preliminary  analyses  showed  that  very  good 
duplicates  could  be  obtained  by  this  procedure. 

In  the  rabbits,  simultaneous  determinations  were  made  of  the 
water,  ether-extract,  and  ash  of  the  muscles.  Since  only  slight 
variations  in  the  percentages  of  ether-extract  and  ash  occurred, 
these  estimations  were  omitted  in  the  fowl.  In  the  rabbits  the 
creatine  content  is  calculated  on  the  moist  muscle,  and  on  the 
ether-extract-  and  fat-free  dry  material;  while  in  the  fowl  the  per- 
centages are  calculated  on  the  bases  of  moist  and  dry  tissue. 

The  amount  of  water  in  the  normal  tissues  is  reasonably  con- 
stant. The  higher  figure  obtained  with  rabbit  4  is  due  to  the 
fact  that  the  animal  had  received  an  intravenous  injection  of 
150  cc.  of  dilute  adrenalin  solution,  in  connection  with  other 
investigations.  Only  a  small  amount  of  the  injected  water  had 
been  excreted  when  death  occurred.  In  the  starving  animals, 
the  water  content  progressively  increased  as  starvation  was  pro- 
longed. Similar  results  were  obtained  by  v.  Boethlingk1  on  mice. 
This  increase  in  water  may  have  caused  the  apparent  decrease 
in  the  amount  of  creatine  observed  by  Dorner,  and  calculated  by 
him  on  the  moist  material. 

The  ether-extract  is  also  constant  in  the  normal  animals,  and 
shows  a  decrease  during  fasting.  The  figures  agree  with  those 
found  in  starving  rabbits  by  Rubner.2  This  author  reports  that 
in  starvation  the  muscles  of  rabbits  contain  2  to  3  per  cent  of  fat 
calculated  on  the  dry  material.  Assuming  that  the  muscles  of 
his  animals  contained  approximately  80  per  cent  of  water,  his 
figures  agree  with  those  reported  in  Table  II.  The  percentage 
of  fat  in  the  well-fed  rabbits  is  somewhat  lower  than  that  reported 

*v.  Boethlingk:    Arch.  d.  scienc.  biol.,  v,  p.  395,  1897. 
2 Rubner:    Zeitschr.  f.  Biol.,  xvii,  p.  229,  1881. 


Lafayette  B.  Mendel  and  William  C.  Rose  259 


for  the  wild  hare  by  Konig  and  Farwick,1  who  found  1.07  per  cent 
of  ether-extract  in  the  muscles  of  the  extremities.  Moreover, 
the  fat  content  of  other  animals  is  usually  higher  than  that  of 
the  rabbit.  According  to  the  analyses  of  Almen,2  the  muscles 
of  lean  oxen  contain  1.5  per  cent  of  fat  and  76.7  per  cent  of  water, 
while  the  muscles  of  pigeons  contain  1.0  per  cent  of  fat.3  This 
is  in  accord  with  the  well  known  fact  that  rabbits  usually  have 
relatively  little  subcutaneous  adipose  tissue.  They  seem  to  store 
fat  only  with  difficulty. 


table  1. 

The  creatine  content  of  muscle. 


AUTHOR 

REFERENCE 

ANIMAL 

CONDITION  OP 
MUSCLE 

CREATINE 
IN  MOIS1 
TISSUE 

Mellanby  

Journ.  Physiol.,  xxxvi,  p. 

per  cent 

472,  1907-8 

Frog 

Normal 

0.302 

Graham- 
Brown  and  » 

Biochem.   Journ.,  iv,  p. 

Frog 

Normal 

0.377 

Cathcart 

A  01  IOAO 

Frog 

Isolated  and 

0.413 

stim. 

Mellanby  

Journ.  Physiol,  xxxvi,  p. 

472,  1907-8 

Fowl 

Normal 

0.360 

Mellanby  

Journ.  Physiol.,  xxxvi,  p. 

460,  1907-8 

Rabbit 

Normal  (leg) 

0.520 

Mellanby  

Journ.  Physiol.,  xxxiv,  p. 

460,  1907-8 

Rabbit 

Normal  (back) 

0.505 

Mellanby  

Journ.  Physiol.,  xxxiv,  p. 

460,  1907-8 

Rabbit 

Stimulated 

0.506 

Dorner  

Zeitschr.  f.  physiol.  Ch., 

lii,  p.  265,  1907 

Rabbit 

Normal 

0.529 

Dorner  

Zeitschr.  f.  physiol.  Ch., 

lii,  p.  265,  1907 

Rabbit 

Normal 

0.496 

Dorner  

Zeitschr.  f.  physiol.  Ch., 

lii,  p.  265,  1907 

Rabbit 

Normal 

0.496 

Dorner  

Zeitschr.  f.  physiol.  Ch., 

lii,  p.  265,  1907 

Rabbit 

Normal 

0.505 

Dorner  

Zeitschr.  f.  physiol.  Ch., 

iii,  p.  265,  1907 

Rabbit 

Starving 

0.414 

1  Konig  and  Farwick:    Zeilsch'.  f.  Biol.,  xii,  p.  497,  1876. 
2Alm6n:    Nova  Act.  Reg.  Soc.  Scient.  Upsal.,  vol.  extr.  ord.,  1877. 
3Cf.  Konig:    Chem.  d.  menschl.  Nahrungs-  u.  Genussmittel,  i,  p.  42,  1903 


Creatine  and  Creatinine  Metabolism 


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Lafayette  B.  Mendel  and  William  C.  Rose  261 


The  amount  of  ash  undergoes  very  little  change  in  rabbits 
during  fasting.  The  figures  compare  favorably  with  those  found 
by  Konig  and  Krauch,1  the  average  of  which  was  1.17  per  cent. 

Creatine  content.  A  summary  of  the  more  important  records 
of  the  creatine  content  of  muscle  is  given  in  Table  I.  Where  these 
figures  were  expressed  as  creatinine  in  the  original  papers,  they 
have  been  recalculated  by  us  and  expressed  as  creatine.  A  com- 
parison of  these  data  with  our  analyses  will  show  a  good  agree- 
ment in  the  creatine  content  of  muscle  in  so  far  as  normal,  i.e., 
fed  animals  are  concerned. 

TABLE  III. 


Creatine  in  starving  hens'  muscle. 


£4 

MUSCLE  ANALYSES 

NUMBER  OF 
ANIMAL 

DURATION  OF 
STARVATION 

INITIAL  WEIGH' 

FINAL  WEIGHT 

LOSS  IN  WEIGH' 

LOSS  IN  WEIGH' 

Water 

Muscle  used 
for  creatine 
estimated 

Creatine  re- 
covered 

Creatine  In 
moist  muscle 

Creatine  in 
dry  muscle 

days 

grams 

grams 

grams 

percent 

percent 

grams  |  gram 

percent 

percent 

14 

0 

1750 

1750 

0 

0 

73.00 

41.34  0.170 

0.411 

1.52 

15 

0 

1890 

1890 

0 

0 

73.05 

41.08  0.170 

0.414 

1.54 

16 

11 

2160 

1775 

385 

17.8 

73.31 

41.25  0  181 

0.439 

1.64 

17 

19 

2260 

1845 

415 

18.4 

73.66 

40. 4C!  0.200 

0.495 

1.88 

18 

11 

2160 

1710 

450 

20.8 

73  24 

41  45  0.184 

0.444 

1.66 

19 

17 

1170 

820 

350 

30.0 

76.27 

22.61  0.087 

0.384 

1.62 

20 

19 

1520 

1000 

520 

34.2 

76.85 

32.12  0.138 

0.430 

1.85 

The  data  obtained  in  the  present  investigation  of  starving 
animals,  on  the  other  hand,  exhibit  distinctly  higher  values. 
There  are  few  comparable  experiments  on  record.  The  protocols 
are  arranged  in  the  Tables  (II  and  III)  in  the  order  of  the  progres- 
sive percentage  loss  in  weight  of  the  animals.  "With  a  single  excep- 
tion (rabbit  12)  the  muscles  of  all  starving  animals  showed  a  higher 
percentage  of  creatine  than  the  fed  controls.  The  increase  in 
creatine  tends  to  be  proportional  to  the  percentage  loss  in  weight 
of  the  animals.  No  explanation  can  be  given  for  the  isolated  low 
figures  obtained  with  animal  12.  The  difference  may  have  been 
due  in  part  to  age,  for  Mellanby  ('07-08)  has  shown  that  consider- 
able variation  in  the  creatine  content  is  associated  with  this  fac- 
tor.   Young  animals  have  much  less  creatine  than  adults. 

1  Konig  and  Krauch:  Che?n.  d.  menschl.  Nahrungs  n.  Genussmittel,  i,  p. 
40,  1903. 


262         Creatine  and  Creatinine  Metabolism 


In  the  experiments  with  hens  the  results  are  comparable  to  those 
obtained  on  rabbits.  Both  water  and  creatine  increased  in  per- 
centage as  starvation  was  prolonged.  In  fowl  19  the  creatine 
calculated  on  the  moist  muscle  is  lower  than  in  the  control  birds, 
but  when  expressed  on  the  basis  of  the  dry  tissue,  is  slightly  higher 
than  the  controls.  The  low  figures  here  are  without  doubt  due 
to  age,  for  this  fowl  was  small  and  immature. 

It  is  noticeable  that  the  increases  in  creatine  in  the  fowl  are  not 
as  large  as  in  the  rabbits.  Most  of  the  former  were  in  good  nutri- 
tive condition  when  killed.  All  but  the  half-grown  hen  (No.  19) 
had  considerable  subcutaneous  fat.  Since  they  retain  fat  so  read- 
ily, it  may  be  equally  true  that  they  retain  considerable  glycogen, 
and  a  marked  increase  in  creatine  would  not  be  expected.  It  is 
certainly  true  that  as  a  rule  fowl  can  fast  much  longer  than  rab- 
bits before  death  results. 

DISCUSSION. 

Since  a  significant  and  progressive  increase  in  creatine  has  been 
demonstrated  in  muscle  during  inanition,  there  seems  to  be  no 
reasonable  ground  for  doubting  the  origin  of  urinary  creatine  and 
creatinine  in  endogenous  metabolism.  These  results  are  in  strik- 
ing contrast  to  those  reported  for  a  dog  by  Howe  and  Hawk  ('11). 
They  (toe.  cit.,  p.  239)  estimated  that  "the  amount  of  creatine 
nitrogen  present  in  the  muscles  of  the  dog  at  the  end  of  the  fast 
was  0.042  per  cent,  showing  a  very  marked  decrease  (66  per  cent)." 
It  is  not  apparent  in  the  preliminary  report  of  Howe  and  Hawk 
whether  this  finding  was  duplicated  or  not.  In  order  to  prove 
conclusively  such  a  remarkable  reduction,  it  would  be  necessary 
to  have  data  on  a  number  of  animals  of  approximately  the  same 
age  as  the  controls. 

Howe  and  Hawk  believe  that  the  pronounced  decrease  in  crea- 
tine is  "a  most  significant  fact  and  shows  clearly  that  in  fasting 
we  cannot  with  accuracy  consider  the  total  amount  of  excreted 
creatine  as  resulting  from  the  complete  and  permanent  disintegra- 
tion of  muscular  tissue."  Again  they  say,  "a  large  part  of  the 
creatine  excreted  during  the  fasts  and  which  is  ordinarily  considered 
as  representing  completely  disintegrated  muscular  tissue,  in  reality 
most  certainly  does  not  represent  this  but  rather  has  been  with- 
drawn from  muscular  tissue  which  is  still  functioning  as  living 
tissue  within  the  body  of  the  animal."    It  is  difficult  to  under- 


Lafayette  B.  Mendel  and  William  C.  Rose  263 


stand  how  any  such  withdrawal  of  creatine  from  functioning  tis- 
sue could  occur.  Urano  ('07)  has  shown  that  muscle  creatine  is 
held  in  a  non-diffusible  form  and  is  probably  loosely  combined  with 
the  muscle  protoplasm.  Its  liberation  would  only  be  possible 
after  complete  disintegration  of  the  muscle  bundles. 

As  evidence  that  creatine  is  not  an  index  of  muscle  disintegra- 
tion, Howe  and  Hawk  point  to  the  discrepancy  between  the 
amounts  of  muscle  catabolism  calculated  on  the  two  bases  of  total 
nitrogen  and  creatine  excretion.  They  find  that  only  about  one- 
half  of  the  urinary  nitrogen  can  be  accounted  for,  when  the  tissue 
catabolism  is  calculated  on  the  creatine  basis.  Several  sources 
of  error  are  apparent  in  such  a  calculation.  The  authors  seem  to 
assume  that  all  urinary  nitrogen  has  its  origin  in  muscle  catabolism. 
It  is  true  that  muscle  represents  the  major  part  of  the  body  tis- 
sues, and  that  it  undergoes  a  great  loss  in  weight  during  inanition ; 
but  Voit1  has  shown  that  the  glandular  tissue  undergoes  much 
greater  loss  in  weight  than  does  muscle.  Creatine  may  be  a  prod- 
uct of  the  metabolism  of  glandular  tissue;  but  with  our  present 
knowledge  there  is  certainly  no  evidence  for  such  an  assumption. 
Moreover,  dogs  invariably  have  an  enormous  store  of  reserve 
nitrogen,  which  might  increase  the  output  of  total  nitrogen  dur- 
ing starvation  without  altering  the  creatine  excretion.  But  the 
greatest  fallacy  in  the  reasoning  of  Howe  and  Hawk  is  that  they 
entirely  neglect  to  consider  the  output  of  preformed  creatinine  in 
their  calculations,  and  apparently  assume  for  it  an  origin  distinct 
from  that  of  creatine.  If  the  tissue  catabolism  is  calculated  on 
the  basis  of  the  output  of  total  creatinine  (i.e.,  creatine  plus  creat- 
inine), the  discrepancy  between  the  figure  so  obtained,  and  that 
obtained  on  the  total  nitrogen  basis,  will  be  found  to  be  much 
smaller.  Indeed,  in  the  second  fasting  period  of  Howe  and  Hawk's 
dog,  the  muscle  waste  calculated  from  the  total  creatinine,  is  more 
than  sufficient  to  account  for  all  urinary  nitrogen. 

All  methods  of  calculating  tissue  loss  on  this  basis  must  be 
inaccurate.  The  catabolism  of  tissues  other  than  muscle  tissue; 
the  excretion  of  reserve  nitrogen;  the  possibility  of  a  resynthesis 
of  nitrogen  (cf.  Paton,  '09-10);  changes  in  the  rate  of  formation, 
oxidation,  retention,  and  excretion  of  creatine  and  creatinine; 
all  tend  to  render  such  calculation  inconclusive.    The  fact  that 

lVoit:   Hermann's  Handbuch,  vi,  part  i,  pp.  96-97. 


264         Creatine  and  Creatinine  Metabolism 


calculations  dependent  upon  so  many  variables  do  not  exactly 
agree,  is  not  surprising.  It  would  rather  be  surprising  if  they  did 
agree.  Hence,  by  these  methods  no  evidence  can  be  obtained 
which  would  invalidate  the  hypothesis  that  creatine  and  creatin- 
ine are  products  of  the  endogenous  metabolism  of  muscle  tissue. 

Without  doubt  there  occurs  an  increase  in  the  percentage  of  cre- 
atine in  the  muscles  of  rabbits  and  fowl  during  inanition.  Two 
possible  explanations  for  the  larger  amount  of  creatine  suggest 
themselves.  The  increase  may  be  due  (1)  to  a  removal  of  the 
non-creatine  portion  of  the  muscle,  leaving  the  creatine  intact; 
or  (2)  to  an  increased  formation  of  creatine.  The  first  explana- 
tion seems  improbable,  for  if  the  creatine  is  loosely  combined  with 
muscle  as  Urano  ('07)  believes,  there  is  no  reason  for  assuming 
that  the  non-creatine  material  could  be  withdrawn  and  leave  the 
creatine  intact,  any  more  than  for  assuming  that  the  reverse  could 
occur.  An  increased  formation  seems  to  be  the  most  plausible 
explanation.  This  view  is  in  accord  with  the  numerous  obser- 
vations already  cited  in  a  former  paper  (Mendel  and  Rose,  '11). 
During  such  an  accelerated  production  of  creatine,  it  would  prob- 
ably be  liberated  in  part  by  the  muscles  and  appear  in  the  urine, 
for  the  muscles  would  become  supersaturated  and  be  unable  to 
combine  with  all  of  the  excess. 

BIBLIOGRAPHY. 

Demant:    Zeitschr.f.  physiol.  Chem.,  iii,  pp.  381-90,  1879. 
Dorner:   ibid.,  Iii,  pp.  225-78,  19C7. 

von  Furth  and  Schwarz:    Biochem.  Zeitschr.,  xxx,  pp.  413-32,  1911. 
Graham-Brown  and  Cathcart:  J 'our -n.  of Physiol.,  xxxah,  p.  xiv,  1908. 
Graham-Brown  and  Cathcart:   Biochem.  Journ.,  iv,  pp.  420-26,  1909. 
Grindley  and  Woods:    Journ.  of  Biol.  Chem.,  ii,  pp.  309-15,  1906-07. 
Howe  and  Hawk:    Journ.  Amer.  Chem.  Soc.,  xxxiii,  pp.  215-54,  1911. 
Liebig:   Annal.  d.  Chem.  u.  Pharm.,  lxii,  pp.  257-369,  1847. 
Mellanby:    Journ.  of  Physiol.,  xxxvi,  pp.  447-87,  1908. 
Mendel  and  Rose:   Journ.  of  Biol.  Chem.,  x,  p.  213,  1911. 
Monari  :   Atti.  R.  Accad.  d.  Sc.  d.  Torino,  xxii,  pp.  846-64,  1887. 
Abstr.,  Maly's  Jahresb.,  xvii,  p.  311-12,  1887. 

Nawrocki:    Zeitschr.f.  analyt.  Chem.,  iv,  pp.  330-48,  1865. 
Paton:    Journ.  of  Physiol.,  xxxix,  pp.  485-504,  1909-10. 
Sarokow:    Virchow's  Arch.,  xxviii,  pp.  544-51,  1863. 
Sczelkow:    Centralb.  f.  d.  med.  Wiss.,  p.  481,  1866. 
Urano:    Hofmeister's  Beitrdge,  ix,  pp.  104-15,  1907. 
Voit:    Zeitschr.  f.  Biol.,  iv,  pp.  77-93,  1868. 
Weber:    Arch.  f.  exp.  Path.  u.  Pharm.,  lviii,  pp.  93-112,  19C7. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  X,  No.  3,  1911 


EXPERIMENTAL  STUDIES  ON  CREATINE  AND 
CREATININE. 

in.  EXCRETION  OF  CREATINE  IN  INFANCY  AND  CHILDHOOD.1 

By  WILLIAM  C.  ROSE. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  August  14,  1911.) 

Physiological  literature  contains  scarcely  any  data  on  the 
creatine-creatinine  metabolism  of  children.  Before  the  introduc- 
tion of  the  Folin  method  for  the  estimation  of  these  substances, 
creatinine  was  supposed  to  be  entirely  absent  from  the  urine  of 
the  young.  Rietschel  ('05)  was  unable  to  detect  it  in  normal 
suckling  infants  by  the  Salkowski-Neubauer  method,  or  by  the 
color  reaction  of  Weyl.  Van  Hoogenhuyze  and  Verploegh  ('05), 
Amberg  and  Morrill  ('07),  Funaro  ('08),  Amberg  and  Rowntree 
('10),  and  Sedgwick  ('10),  using  the  Folin  method,  have  now  con- 
clusively demonstrated  that  creatinine  is  a  constant  constituent 
of  the  urine  of  sucklings.  All  investigators  find  the  creatinine 
excretion  to  be  very  low,  ranging  usually  from  6  to  10  mgms.  per 
kilo  of  body  weight. 

Assuming  the  origin  of  urinary  creatine  and  creatinine  to  be  muscle 
creatine,  one  would  expect  to  find  creatinine  in  the  urine  of  sucklings. 
Mendel  and  Leavenworth  ('08)  have  shown  that  the  embryonic 
muscles  of  the  pig  contain  an  average  creatine  content  of  0.03  per 
cent  of  the  moist  tissue.  An  analysis  made  by  the  writer  of  the 
muscles  of  a  new  born  infant  (see  Table  I)  indicates  that  creatine 
is  present  in  somewhat  larger  amount  than  in  embryonic  muscle, 
though  still  far  less  than  the  amount  present  in  the  muscles  of 
an  adult.    These  observations  are  in  accord  with  those  of  Mel- 

xThe  data  are  taken  from  the  thesis  presented  by  the  author  for  the  degree 
of  Doctor  of  Philosophy,  Yale  University,  1911. 

265 


266 


Creatine  and  Creatinine  Metabolism 


lanby  ('08) — that  the  creatine  content  of  muscle  greatly  varies 
with  the  age  of  the  animal.  They  serve  also  to  throw  some  light 
on  the  extremely  low  creatinine-coefficients  of  sucklings,  and  fur- 
nish further  evidence  for  the  origin  of  urinary  creatinine  in  mus- 
cle metabolism. 


TABLE  I. 

Analysis  of  the  muscle  of  a  new-born  infant. 


WATER 

ASH 

ETHER 

MUSCLE 
USED  FOR 

CREATINE 

CREATINE 
IN  MOIST 
MUSCLE 

CREATINE  IN 
WATER-,  ASH-, 

AND  ETHER 
EXTRACT-FREE 

MUSCLE 

EXTRACT 

CREATINE 

FOUND 

ESTIMATION 

per  cent 

per  cent 

per  cent 

grams 

gram 

per  cent 

per  cent 

80.12 

0.69 

5.94 

37.03 

0.072 

0.19 

1.46 

Amberg  and  Morrill  ('07)  found  creatine  to  be  present  in  the 
urine  of  a  single  infant  tested  by  them.  Similar  observations  were 
made  on  sucklings  by  Sedgwick  ('10)  and  van  Hoogenhuyze  and 
ten  Doeschate  ('11),  and  on  young  puppies  and  kittens  by  Clos- 
son  ('06).  In  one  experiment  on  a  puppy  three  weeks  old,  Clos- 
son  found  18  mgms.  of  total  creatinine  to  be  excreted  per  day, 
of  which  17  mgms.  were  in  the  form  of  creatine.  At  the  age  of 
eight  and  a  half  weeks,  the  total  creatinine  excretion  per  day  was 
48  mgms.,  of  which  29  mgms.  were  in  the  form  of  creatine. 

The  data  regarding  the  elimination  of  creatine  in  children  more 
than  a  year  of  age,  are  exceedingly  scanty.  Schwarz  ('10)  reported 
that  a  normal  five-year-old  boy  examined  by  him  excreted  no 
creatine,  while  rachitic  children  of  the  same  age  excreted  30  to  50 
per  cent  of  their  total  creatinine  in  the  form  of  creatine. 

In  view  of  the  observations  that  creatine  is  a  normal  constitu- 
ent of  the  urine  of  sucklings,  it  was  of  interest  to  determine  the  age 
at  which  this  product  disappeared.  Numerous  specimens  of  urine 
from  children  of  different  ages  were  analyzed,  the  result  of  which 
are  summarized  in  Tables  II  and  III.  Since  it  was  impractical 
to  collect  the  complete  excretion  for  twenty-four  hour  periods, 
the  analytical  figures  are  expressed  in  milligrams  per  one  hun- 
dred cubic  centimeters  of  urine.  Specimen  24  was  obtained  from 
a  child  convalescing  after  an  attack  of  mumps;  all  other  samples 
were  obtained  from  apparently  perfectly  normal  individuals. 


William  C.  Rose 


267 


TABLE  11. 


Creatine  in  the  urine  of  male  children.    Amount  in  100  cc. 


TJRINE 

NUMBER 
OF 

AGE 
OP 

0 

a  « 
0  3 

crea- 
ne 

med 

Creatine  as 
creatinine 

ae  In 
nt  of 

REMARKS 

SAMPLE 

CHILD 

Speclfi 
gravlt 

Reactl 
lltm 

Total 
tlnl 

Prefor 
creati 

Creatli 
per  ce 
total 

years 

mgms. 

mams. 

mgms. 

1 

li 

1  .030 

Acid 

110 

00 
88 

OA  A 

20.0 

Perfectly 
normal. 

2 

4 

1 .021 

Acid 

OD 

A*7 

47 

19 

00  0 
28.8 

Perfectly 
normal. 

3 

5 

1.025 

Acid 

66 

50 

16 

24.2 

Perfectly 
normal. 

4 

5 

1.017 

Acid 

159 

33 

126 

79.2 

Sample  from 
same  child  as 
No.  3. 

5 

10 

1  .025 

Acid 

y2 

75 

17 

18.5 

Perfectly 
normal. 

6 

10 

Acid 

75 

a.c\ 
DO 

15 

OA  A 

2U.0 

Perfectly 
normal. 

7 

10 

1 .020 

Acid 

ta 

70 

70 

0 

A 
0 

Perfectly 
normal. 

8 

11 

1.023 

Acid 

113 

96 

17 

1  r  A 

15.0 

Perfectly 
normal. 

9 

11 

1.022 

A     •  J 

Acid 

135 

116 

19 

14. 1 

Protein  present. 

10 

12 

1.025 

Acid 

156 

100 

56 

35.9 

Perfectly 
normal. 

11 

13 

1.019 

Acid 

81 

74 

7 

8.6 

Perfectly 
normal. 

12 

14 

1 .020 

Acid 

68 

49 

19 

OT  A 

27.9 

Perfectly 
normal. 

13 

15 

Acid 

87 

65 

22 

25.3 

Perfectly 
normal. 

14 

17 

1.022 

Acid 

133 

133 

0 

0 

Perfectly 
normal. 

15 

18 

1.028 

Acid 

121 

121 

0 

0 

Perfectly 
normal. 

16 

18 

1.030 

Acid 

270 

270 

0 

0 

Perfectly 
normal. 

17 

18 

1.028 

Acid 

175 

175 

0 

0 

Perfectly 
normal 

18 

19 

1.024 

Acid 

144 

144 

0 

0 

Perfectly 
normal. 

19 

19 

1.018 

Acid 

112 

112 

0 

0 

Perfectly 

normal. 

268        Creatine  and  Creatinine  Metabolism 

TABLE  III. 


Creatine  in  the  urine  of  female  children.    Amount  in  100  cc. 


URINE 

NUMBER 

AGE 

o 

u  g 

OF 

OF 

o  >> 

o  B 

gg 

«  d 

si 

REMARKS 

SAMPLE 

CHILD 

React! 
litm 

— .  d 

o— <  d 

Tota 
fcl 

Pref( 

Crea 
crea 

Crea 
per 

tlni 

years 

mgms. 

mgms. 

mgms. 

20 

n 

1.010 

Acid 

ID 

14. 

o 

19 

1^ .  0 

Perfectly  nor- 
mal child. 

21 

ii 

1.005 

Acid 

17 

1  4 

7 

10 

Oo .  o 

Perfectly  nor- 
mal child. 

22 

3 

1.030 

Neu- 
tral 

DO 

38 
uo 

1  ^ 

9R  3 

Perfectly  nor- 
mal child. 

23 

3 

1.018 

Acid 

68 

40 

28 

41.2 

Perfectly  nor- 
mal child. 

24 

5 

1.030 

Alka- 
line 

72 

60 

12 

16.7 

Mumps. 

25 

7 

1.019 

Acid 

61 

27 

34 

55.7 

Perfectly  nor- 
mal child. 

-26 

7 

1.020 

Acid 

66 

50 

16 

24.2 

Sample  from 
same  child  as 
No.  25. 

27 

7 

1.024 

AlL-o 

AiKa- 
line 

fiO 

oo 

7 

11  7 

Perfectly  nor- 
mal child. 

28 

8 

1.021 

Acid 

4.1 

7 

17  1 

1/  .  1 

Perfectly  nor- 
mal child. 

29 

8 

1.030 

Acid 

on 
yu 

Oo 

Q9 

OO .  D 

Sample  from 
same  child  as 
No.  28. 

30 

11 

1.913 

Acid 

64 

52 

12 

18.8 

Perfectly  nor- 
mal child. 

31 

11 

1.020 

Acid 

7Q 

/y 

oy 

in 

1U 

19  7 

Perfectly  nor- 
mal child. 

32 

12 

1.020 

Acid 

91 

62 

29 

31.9 

Minute  trace  of 
protein. 

33 

13 

1.025 

Acid 

91 

91 

0 

0 

Perfectly  nor- 
mal child. 

34 

13 

1.016 

Acid 

60 

49 

11 

18.3 

Perfectly  nor- 

m  q  1  pVnlH 

35 

13 

1.022 

Acid 

117 

86 

31 

26.5 

Perfectly  nor- 
mal child. 

36 

15 

1.023 

Acid 

83 

61 

22 

26.5 

Trace  of  pro- 
tein present. 

37 

15 

1.016 

Acid 

72 

60 

12 

16.7 

Same  as  36. 
Protein  present. 

38 

20 

1.014 

Acid 

69 

69 

0 

0 

Normal. 

39 

21 

1.016 

Acid 

81 

81 

0 

0 

Normal. 

William  C.  Rose 


269 


The  urines  were  preserved  with  toluene,  and  analyzed  within 
twenty-four  hours  after  excretion.  Except  in  the  cases  indicated 
(Nos.  9,  32,  36  and  37),  protein  and  sugar  were  entirely  absent. 

Contrary  to  the  findings  of  Schwarz,  children  of  five  years  and 
over  excrete  considerable  creatine.  Indeed,  with  the  exception 
of  two  cases,  creatine  was  present  in  all  specimens  from  children 
under  fifteen  years  of  age.  A  boy  of  ten  and  a  girl  of  thirteen 
failed  to  have  creatine  in  their  urines. 

No  progressive  decrease  in  the  percentage  of  the  total  creatinine 
in  the  form  of  creatine  coincident  with  increase  in  age  is  apparent; 
nor  is  the  percentage  of  creatine  constant  for  the  same  individual. 
For  instance,  one  specimen  (No.  3)  obtained  from  a  child  of  five 
years  contained  24.2  per  cent  of  the  total  creatinine  in  the  form  of 
creatine,  while  a  second  sample  (No.  4)  from  the  same  child  a  few 
days  later,  contained  79.2  per  cent  of  the  total  creatinine  as  creatine. 

It  was  impossible  to  obtain  information  as  to  the  amount  and 
kind  of  food  eaten  by  the  children.  Most  of  the  specimens  were 
obtained  from  the  city  orphans  home  or  from  private  families, 
and  the  subjects  of  the  experiments  were  probably  ingesting  more 
or  less  meat.  It  is  possible,  therefore,  that  the  oxidation  or  con- 
version of  creatine  into  creatinine  may  be  difficult  for  young  indi- 
viduals to  accomplish,  and  in  this  case  the  creatine  of  the  urine 
may,  in  part,  represent  ingested  creatine;  or  the  glycogenic  func- 
tions may  be  imperfectly  developed,  and  the  store  of  carbohy- 
drates be  insufficient  to  exert  its  regulatory  influence  over  me- 
tabolism during  childhood.  Frank1  has  shown  that  the  percentage 
of  sugar  in  the  blood  of  infants  is  greater  than  that  of  adults.  It 
is  conceivable  that  the  demand  for  carbohydrates  for  the  histo- 
genetic  processes  may  be  so  great  that  the  cells  are  left  in  partial 
carbohydrate  hunger,  and  are  unable  to  perform  the  "endo-cat- 
abolic,,  activities  as  perfectly  as  in  later  life.  At  any  rate  it  is 
of  some  interest  statistically  to  find  that  creatine  is  usually  pres- 
ent in  the  urine  until  or  after  the  age  of  puberty. 

1  Frank:   Zeitschr.  f.  physiol.  Chem.,  lxx,  pp.  129-42,  1910. 


270        Creatine  and  Creatinine  Metabolism 


BIBLIOGRAPHY. 

Amberg  and  Morrill:    Journ.  Biol.  Chem.,  iii,  pp.  311-20,  1907. 
Amberg  and  Rowntree:    Johns  Hopkins  Hosp.  Bull.,  xxi,  pp.  40-44,  1910. 
Closson:    Amer.  Journ.  of  Physiol.,  xvi,  pp.  252-67,  1906. 
Funaro:    Biochem.  Zeitschr.,  x,  pp.  467-71,  1908. 

van  Hoogenhuyze  and  ten  Doeschate  :    Annal.  d.  gynecol.  et  d'obstet., 
Jan.  et  Fev.,  1911. 

van  Hoogenhuyze  and  Verploegh:    Zeitschr.  f.  physiol.  Chem.,  xlvi, 

pp.  415-71,  1905. 
Mellanby:    Journ.  of  Physiol.,  xxxvi,  pp.  447-87,  1908. 
Mendel  and  Leavenworth:    Amer.  Journ.  of  Physiol.,  xxi,  pp.  99-104, 

1908. 

Rietschel:    Jahrb.  f.  Kinderheilkunde,  lxi,  p.  615,  1905. 

Schwarz:    Ibid.,  lxxii,  pp.  549-74;  712-35,  1910. 

Sedgwick:    Journ.  Amer.  Med.  Assoc.,  lv,  pp.  1178-80,  1910. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  X.  No.  4,  1911 


STUDIES  IN  NUTRITION. 
I.    THE  UTILIZATION  OF  THE  PROTEINS  OF  WHEAT. 

By  LAFAYETTE  B.  MENDEL  and  MORRIS  S.  FINE. 

(From  the  Sheffield  Laboratory  of  Physiological  Chemistry.  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  September  2,  1911.) 

CONTENTS. 


Introduction   303 

Factors  involved   305 

Earlier  studies  with  bread   306 

Earlier  studies  with  isolated  proteins   307 

Methods   308 

Experimental  part   310 

Products  employed   310 

Metabolism  experiments   311 

"Glidin"   311 

Gluten   313 

Glutenin   317 

Gliadin   321 

Nitrogen  balances   324 

Summary   324 


INTRODUCTION. 

For  many  years  unlike  values  in  nutrition  have  been  ascribed 
to  the  proteins  of  animal  and  vegetable  origin.  Now  that  the 
chemical  individuality  and  physiological  specificity  of  the  so- 
called  proximate  principles  are  asserting  their  importance,1  fur- 
ther study  of  the  availability  of  the  foodstuffs  seems  especially 

1  Reference  may  be  made  to  the  recent  researches  on  the  amino-acid 
requirements  of  the  animal  organism  by  Abderhalden,  Henriques,  Michaud, 
(see  bibliography),  and  others.  Cf.  also  the  studies  of  Hunt  (Hygienic 
Laboratory,  Public  Health  and  Marine  Hospital  Service  of  the  United  States, 
Bull.  69,  1910),  which  indicate  that  the  resistance  of  animals  to  certain 
poisons  may  Vary  with  the  character  of  their  diet. 

303 


304 


Utilization  of  Wheat  Proteins 


desirable.  The  opinion  is  freely  expressed  that  the  animal  pro- 
teins are  far  better  utilized  than  those  of  plant  origin,  and  the 
following  statistics  compiled  by  Atwater  and  Bryant1  may  be 
quoted  on  this  point: 


CHARACTER  OF  DIET 


Animal  foods. . . 

Cereals  

Legumes,  dried. 

Vegetables  

Fruits  

Vegetable  foods 
Total  food  


PROTEIN  UTILIZED 


per  cent 

97 
85 
78 
83 
85 
84 
92 


It  can  scarcely  be  said  that  we  are  yet  in  a  position  to  explain 
adequately  the  poorer  utilization  of  the  nitrogen  components  of 
certain  vegetable  foods.  Voit2  and  his  followers3  were  early  aware 
that  structural  peculiarities  of  plant  products,  such  as  cellulose 
walls,  etc.,  render  the  proteins  comparatively  inaccessible  to  the 
digestive  juices,  thus  in  part  explaining  the  possibility  of  poorer 
utilization.  The  question  of  the  relative  availability  and  nutri- 
tive value  of  the  vegetable  proteins  per  se  has  received  little  atten- 
tion, owing  in  large  part  to  the  technical  difficulties  in  securing 
suitable  isolated  products  for  study. 

We  have  undertaken  a  detailed  investigation  of  some  of  the 
factors  involved  in  the  digestibility  and  utilization  of  the  proteins 
of  vegetable  food  materials.  An  attempt  has  been  made  to  elim- 
inate many  of  the  unfavorable  conditions  or  factors  which  attend 
the  use  of  these  plant  products,  and  above  all  to  study  the  nutri- 
tive value  of  their  proteins  as  such.  Incidentally  it  has  become 
necessary  to  devote  some  attention  to  various  features  of  the  ali- 
mentary functions,  such  as  the  origin  of  the  feces,  which  have  an 
important  bearing  on  the  interpretation  of  experimental  results.4 

1  Atwater  and  Bryant :  Report  of  the  Storrs  Agricultural  Experiment 
Station,  1899,  p.  86. 

2  Voit:  Sitzungsberichte  der  Bayerischen  Akademie,  ii  (4),  1869. 
s  Cf.  Rubner:    Zeitschrift  fur  Biologie,  xix,  p.  45,  1883. 

4  The  data  in  this  and  succeeding  papers  of  this  series  are  taken  from  the 
dissertation  of  M.  S.  Fine,  Yale  University,  1911. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  305 


FACTORS  INVOLVED. 

The  low  nitrogen  content  of  most  vegetable  foods  necessitates 
the  ingestion  of  a  relatively  large  volume.  This  generally  in- 
creased bulk  of  vegetable  food  may  of  itself  lead  to  more  rapid  evac- 
uation and  lessen  the  possibilities  of  digestion  and  absorption. 

Again,  in  comparison  with  products  of  animal  origin  the  vege- 
table foods  may  present  an  unfavorable  texture.  In  older  plants 
the  cell  walls  may  be  quite  tough  and  even  supplemented  with 
lignin.  There  is  evidence  that  cellulose  is  not  digested  to  any 
considerable  extent  by  the  higher  animals,1  and  the  vegetable 
membranes  are  not  always  easily  permeable  to  the  digestive  juices.2 
It  is  of  primary  importance  for  digestion  that  the  plant  cells  should 
be  thoroughly  disintegrated.  That  ordinary  cooking  is  not  suffi- 
ciently rigorous  treatment  to  bring  about  complete  rupture,  is 
brought  out  in  a  subsequent  paper  of  this  series.  Observations 
by  Wicke  indicate  that  the  nitrogen  utilization  of  diets  containing 
much  bread  becomes  more  and  more  unfavorable  with  increase 
in  the  cellulose  content.  Thus,  with  an  almost  constant  nitrogen 
intake,  the  nitrogen  utilization  was  79,  75,  63,  69,  53  per  cent 
respectively  with  a  cellulose  concentration  in  the  food  of  0.2,  0.3, 
1.1,  1.3,  1.6  per  cent.  This  condition  is  probably  due  in  great 
part  to  the  incomplete  rupture  of  the  cells.  Whether  the  cellu- 
lose per  se  exerts  an  unfavorable  influence,  that  is  when  it  cannot 
be  accused  of  rendering  the  nutrients  inaccessible  to  the  digestive 
agents,  is  a  question  which  has  received  comparatively  little  atten- 
tion.   The  matter  will  be  discussed  fully  in  a  later  paper. 

Mechanical  factors  may  influence  the  rate  of  passage  through  the 
alimentary  canal.  This  applies  to  coarse  particles  such  as  are 
derived  from  seed  coats  in  bran.  Wheat  bran  and  similar  products 
contain  phytin  to  which  a  laxative  action  has  been  attributed  in 
the  case  of  cattle.3  The  fermentative  development  of  acid  and 
gas  with  the  consequent  stimulation  of  peristalsis  has  likewise 

1  Cf .  Scheimert  and  Lotsch:    Biochemische  Zeitschrift,  xx,  p.  10,  1909. 

2  Cf.  Rubner,  loc.  cit. 

3  Cf.  Jordan,  Hart,  and  Patten:  American  Journal  of  Physiology,  xvi, 
p.  268,  1906;  Hart,  McCollum  and  Humphrey:  Wisconsin  Agricultural 
Experiment  Station,  Research  Bull.  No.  5,  1909;  also  Mendel  and  Underhill: 
American  Journal  of  Physiology,  xvii,  p.  75,  1906. 


306 


Utilization  of  Wheat  Proteins 


been  pointed  out  in  connection  with  utilization.  For  example, 
Menicanti  and  Prausnitz  noted  that  poor  utilization  of  bread  nitro- 
gen is  accompanied  by  high  acidity  of  the  feces. 

In  addition  to  the  preceding  considerations  we  must  ask  our- 
selves whether  the  vegetable  proteins  by  themselves  exhibit  any 
inherent  resistance  to  the  digestive  enzymes  of  man.1  To  this  ques- 
tion we  have  devoted  special  attention. 

EARLIEK  STUDIES  WITH  BREAD. 

The  actual  nutritive  value  of  bread  was  early  investigated  by 
Bischoff  and  Voit  on  dogs.  They  found  the  nitrogenous  constit- 
uents of  bread  to  be  80  to  84  per  cent  available.  E.  Bischoff 
reported  a  more  extended  study,  in  which  the  nitrogen  of  bread 
was  shown  to  be  82  to  85  per  cent  utilized.  When  the  nitrogen 
and  starch  of  bread  were  replaced  by  the  nitrogen  of  meat  and  pure 
starch,  the  utilization  was  92  per  cent,  which  result  would  lead  one 
to  believe  that  the  low  digestibility  of  the  bread  nitrogen  might 
be  attributed  to  the  unfavorable  texture. 

Meyer  also  found  that  the  texture  plays  an  important  role  in 
the  utilization.  "Semmel" — white  bread  made  of  the  finest 
flour — was  80  per  cent  available,  while  "  Pumpernickel' 1  had  a 
digestibility  of  but  58  per  cent.  Rubner  obtained  results  essen- 
tially the  same  as  those  reported  by  Meyer. 

Wicke  found  decorticated  wheat  bread  to  be  more  thoroughly 
utilized  than  undecorticated,  thus  being  in  accord  with  Rubner's 
experiments,  in  which  it  was  shown  that  the  nutritive  value  of 
bread  becomes  lower  as  the  bran  content  increases. 

From  the  studies  of  Menicanti  and  Prausnitz  it  appears  that 
the  nitrogen  of  rye  bread  is  less  digestible  (70  per  cent)  than  that 
of  wheat  (87  per  cent),  while  bread  made  of  equal  parts  rye  and 
wheat  had  a  nutritive  value  between  the  two  (80  to  82  per  cent). 

From  the  data2  thus  briefly  cited,  it  is  apparent  that  the  nitrog- 
enous constitutents  of  products  made  of  decorticated  finely  ground 

1  Cf .  Moore,  in  Schdfer's  Textbook  of  Physiology,  p.  441, 1898,  and  Hammar- 
sten:    Lehrbuch  der  physiologischen  Chemie,  1909. 

2  For  other  experiments,  in  which  bread  formed  a  larger  or  smaller  part 
of  the  diet,  see  the  digest  by  Atwater  and  Langworthy:  Office  of  Exper- 
iment Stations,  Bull.  45,  1897. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  307 


wheat  are  the  most  thoroughly  digested,1  although  evidently 
not  as  completely  as  meat,  whereas  the  coarse  breads,  made  of 
undecorticated  flours,  are  very  poorly  utilized.  Between  these 
there  are  all  gradations,  depending  upon  the  texture.  It  is  wor- 
thy of  note  that  under  apparently  the  same  conditions  of  texture, 
etc.,  rye  bread  is  less  well  utilized  than  wheat  bread. 

In  the  studies  above  reviewed  the  nitrogen  of  bread  has  in  no 
case  been  shown  to  be  as  available  to  the  organism  as  that  of  meat. 
However,  it  is  difficult  to  deduce  satisfactory  conclusions  from  these 
experiments  as  there  has  usually  been  some  complicating  influence 
— bran,  cellulose,  acidity,  etc.  In  the  properly  conducted  exper- 
iment one  would  employ  the  pure  protein,  free  from  bran,  starch, 
and  cellulose,  or  the  latter  at  least  thoroughly  disintegrated.  The 
starch  can  very  easily  be  washed  out  of  flour,  and  thus  a  fairly 
pure  protein  preparation  obtained.  The  resulting  material — 
gluten — is  a  common  commercial  article.  A  similar  product — 
"aleuronat" — is  slightly  changed  gluten. 

EARLIER  STUDIES  WITH  ISOLATED  PROTEINS. 

Rubner  found  the  utilization  of  macaroni  noodles  with  and  with- 
out gluten  to  be  89  and  83  per  cent  respectively.  However,  the 
nitrogen  intake  in  the  former  diet  was  twice  as  great  as  that  in 
the  latter,  and  there  is  thus  the  possibility  that  with  equal  nitro- 
gen intakes  the  coefficients  of  digestibility  would  have  been  more 
nearly  alike.  In  an  experiment  on  a  man  Constantinidi  found 
gluten  to  be  94  per  cent  digested,  and  the  utilization  in  two  exper- 
iments on  a  dog  was  97  per  cent.  Potthast  showed  this  material 
to  be  92  per  cent  available,  and  Lusk  obtained  the  somewhat 
less  favorable  result  of  87  per  cent.  Kornauth  found  the  utiliza- 
tion of  gluten  to  be  91  per  cent  against  78  to  82  per  cent  for  dried 
meat  protein. 

The  thorough  digestibility  of  "  aleuronat"  has  been  demonstrated 
by  many  workers,  notably  Bornstein,  Laves,  Wintgen,  and  Sal- 
kowski.  In  recent  years  gliadin,  among  other  proteins,  has  been 
the  object  of  study,  with  particular  reference  to  its  ability  to  main- 

1  Cf.  Woods  and  Merrill:  Office  of  Experiment  Stations,  Bull.  85,  1900. 
These  authors  give  the  utilization  of  the  protein  of  white  bread  as  86  per 
cent,  being  the  average  of  thirteen  experiments. 


3o8 


Utilization  of  Wheat  Proteins 


tain  nitrogen  equilibrium.  Incidentally  we  may  glean  some  data 
bearing  upon  the  subject  of  protein  utilization.  Abderhalden 
found  gliadin  to  be  94  to  98  per  cent  digested,  this  result  being  even 
better  than  the  utilization  of  90  per  cent  for  horse  meat.  Michaud 
obtained  coefficients  of  digestibility  for  "glidin"  of  86  to  96  per 
cent.  Buslik  and  Goldhaber  also  worked  with  " glidin"  and  found 
its  utilization  to  be  as  good  or  better  than  that  of  meat  nitrogen. 

METHODS. 

The  ideal  method  for  the  elucidation  of  the  question  as  to  the 
relative  degree  of  digestibility  of  animal  and  vegetable  proteins 
would  seem  to  be  the  feeding  of  such  mixtures  of  the  pure  foodstuffs 
protein,  sugar  and  fat — free  from  starch  and  cellulose.1  By  this 
procedure  one  would  avoid  the  complicating  factors  of  excessive 
volume,  characteristic  of  plant  food,  and  the  inaccessibility  of 
the  food  materials  due  to  the  inclusion  of  these  substances  within 
the  impenetrable  cells.  In  some  instances  this  ideal  has  been 
strictly  followed;  in  others  the  cellulose  was  not  removed,  but  the 
plant  cells  were  thoroughly  broken  by  heating  or  grinding  to  an 
impalpable  powder. 

In  general,  for  the  present  experiments,  periods  of  meat  feeding 
were  interposed  between  the  experimental  periods.  The  animals 
were  thus  kept  in  good  condition;  any  disturbing  influence  of  one 
diet  would  probably  be  overcome  before  the  feeding  of  the  next 
food  under  investigation;  and  finally  all  experimental  foods  were 
adequately  controlled  by  the  thoroughly  digested  meat  diets.  The 
fat  content2  of  the  meat  was  not  determined;  hence  it  cannot  be 
stated  to  exactly  what  extent  the  calorific  intakes  in  the  differ- 
ent periods  were  comparable.  One  or  more  of  the  proteins  of 
wheat  supplied  all  the  nitrogen  of  the  diet.  The  nitrogen  intakes 
were  practically  the  same  over  long  periods  of  time;  when  for  any 
reason  the  nitrogen  intake  was  changed,  a  preliminary  period  of 
two  to  three  days  always  preceded  the  period  of  actual  observa- 
tion on  the  new  nitrogen  level,  thus  giving  an  opportunity  for 
readjustment. 

1  For  criticism  of  this  viewpoint  cf.  Bryant  and  Milner  :  American 
Journal  of  Physiology,  x,  p.  84,  1903. 

2  Arbitrarily  assumed  to  be  10  per  cent. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  309 


The  feces  accruing  from  the  various  diets  were  identified  by 
giving  a  capsule  of  lampblack  or  carmine1  with  the  first  meal  of 
each  period.  Unless  dry  when  collected,  the  feces  were  preserved 
in  acidified  alcohol  until  all  the  feces  of  the  period  had  been  assem- 
bled, whereupon  they  were  dried  on  the  water  bath  and  finally 
ground  and  analyzed.2 

Current  discussion3  would  seem  to  indicate  that  this  process  of 
drying  on  the  water  bath  occasions  a  loss  of  nitrogen.  The  error 
incident  to  this  procedure,  however,  did  not  appear  to  us  to  warrant 
serious  attention,  at  least  not  until  certain  details  of  metabolism 
operations,  such,  e.  g.,  as  the  accurate  division  of  feces  belonging 
to  successive  periods,  reach  a  higher  stage  of  perfection. 

The  feces  have  only  occasionally  been  diarrhoeal  and  the  ani- 
mals have  never  been  observed  to  dispose  of  their  excrement.  In 
many  instances  agar  agar  or  bone  ash  or  both  of  these  indigestible 
materials  were  added  to  the  diet.  This  served  to  offset  the  diffi- 
culty in  obtaining  satisfactory  separations  of  successive  periods 
due  to  infrequent  defecations.  On  the  other  hand  the  objection 
may  very  properly  be  made  that  the  addition  of  these  materials 
unfavorably  influences  the  protein  utilization.  Indeed,  the  experi- 
ments of  Lothrop,4  and  also  some  of  our  own  studies,  amply  confirm 
the  validity  of  these  objections.  Lothrop's  work  shows,  for  exam- 
ple, that  the  addition  to  a  meat  diet  of  1  gram  of  bone  ash  per  kilo  of 
body  weight  may  almost  double  the  nitrogen  loss  through  the  feces. 
However,  it  seems  reasonable  to  suppose  that  the  utilization  of 
all  diets  would  be  similarly  influenced;  and  thus  the  results  for 
various  periods  would  still  be  comparable.  The  actual  specific 
influence  of  these  indigestible  materials  will  be  discussed  in  a 
subsequent  paper. 

1  Each  capsule  contained  an  average  of  0.35  gram  carmine =0.02  gram 
nitrogen— a  negligible  quantity. 

2  The  weights  of  the  feces  and  the  percentage  nitrogen  composition  are 
based  upon  the  air-dried  specimens.  The  comparisons  of  these  values 
which  will  be  frequently  made  throughout  this  work  are,  nevertheless, 
considered  permissible  since  variations  due  to  this  cause  would  undoubt- 
edly be  smaller  than  incidental  variations  from  other  causes. 

3  Cf.  Howe,  Rutherford,  and  Hawk:  Journal  of  the  American  Chemi- 
cal Society,  xxxii,  p.  1683, 1910.  For  the  literature  see  Emmet  and  Grindley : 
ibid.,  xxxi,  p.  569,  1909. 

4  Lothrop:  American  Journal  of  Physiology,  xxiv,  p.  297,  1909. 


Utilization  of  Wheat  Proteins 


EXPERIMENTAL  PART. 

Products  Employed. 

I.  "Glidin"1  a  commercial  preparation.  This  material  is  a 
slightly  yellowish  and  tasteless  white  powder,  which,  according  to 
Bergell,2  and  Thiemer3  is  prepared  from  wheat  flour  by  a  process 
of  washing  and  centrifuging. 

II.  Gluten,4  a  commercial  preparation  manufactured  by  the  Kel- 
logg Food  Company,  of  Battle  Creek,  Mich.  It  consisted  of  thin, 
flat,  yellow  scales,  approximately  yq  inch  in  diameter. 

III.  Glutenin.5  The  specimen  used  in  these  trials  was  prepared 
as  follows:  Wheat  flour  was  washed  thoroughly  with  water  to 
remove  the  starch.  The  resulting  gluten  was  then  extracted  four 
times  with  70  per  cent  alcohol,  i.  e.,  until  the  extracts  were  color- 
less, and  practically  all  gliadin  was  removed.  The  crude  glutenin 
thus  obtained  was  dried,  finely  ground,  extracted  once  with  ether, 
and  finally  ground  to  an  impalpable  powder.  It  was  not  dissolved 
in  alkali  and  reprecipitated  with  acid,  and  hence  still  contained 
practically  all  the  cellulose  of  the  original  wheat  flour.  On  the 
other  hand,  the  possibility  of  the  protein  being  changed  by  solu- 
tion in  alkali  was  avoided. 

IV.  Gliadin.6  The  70  per  cent  alcoholic  extract  obtained  in 
preparing  glutenin,  as  described  above,  was  concentrated  to  a  thick 
syrup,  and  precipitated  by  pouring  into  water  containing  a  little  salt. 
This  glue-like  material  was  dissolved  in  alcohol,  whose  final  strength 
was  70  per  cent,  and  this  time  precipitated  by  pouring  into  95 
per  cent  alcohol.  The  material  thus  obtained  was  dried  with 
alcohol  and  ether  and  ground  to  an  impalpable  powder. 

Dr.  T.  B.  Osborne  very  kindly  supplied  us  with  both  glutenin 
and  gliadin  in  sufficient  quantities. 

1  Obtained  from  Menley  and  James,  New  York  City.  The  material 
contained  14.5  per  cent  nitrogen,  and  did  not  give  a  starch  reaction.  Con- 
siderable of  this  preparation  dissolved  in  warm  70  per  cent  alcohol,  repre- 
cipitating  on  pouring  into  cold  water — a  characteristic  behavior  of  gliadin. 

2  Bergell:    Medizinische  Klinik,  No.  41,  p.  1042,  1905. 

3  Thiemer:    Wiener  medizinische  Presse,  No.  47,  p.  2431,  1906. 

4  This  material  was  very  kindly  furnished  by  Dr.  Kellogg.  It  contained 
14  per  cent  nitrogen. 

5  For  description  of  glutenin  and  gliadin,  see  T.  B.  Osborne:  "Die 
Pflanzenproteine,"  Ergebnisse  der  Physiologie,  x,  p.  47,  1910. 

6  Cf.  preceding  footno'e. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  311 


Metabolism  Experiments. 

"Glidin" — Tables  1  to  3.  Detailed  information  on  the  nature 
of  the  food  ingredients  may  be  obtained  from  the  tables.  In  the 
periods  of  meat  feeding,  the  mixture  of  cane  sugar,  lard,  agar  and 
bone  ash  was  heated  on  the  water  bath,  till  the  lard  had  melted, 


TABLE  1. 

'Glidin"  with  Agar  and  Bone  Ash. 


SUBJECT,  DOG  5 

Weight  at  beginning,  5.4  Kg. 
Weight  at  end,  5.2  Kg. 


PERIOD  III 

(4  days) 
Meat  Feeding 


PERIOD  IV 

(4  days) 
"Glidin" 
Feeding 


PERIOD  v 

(5  days) 
"Glidin" 
Feeding 


(4  days) 
Meat  Feeding 


Composition  of  daily 
diet  


Nitrogen  output. 
Urine  nitrogen,  gm.... 

Total  nitrogen,  gm  

Nitrogen  in  food,  gm.. 
Nitrogen  balance,  gm. 

Feces. 
Weight  air  dry,  gm. . . . 

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization, 
per  cent  


grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  3 
Bone  Ash  7 
Water  100 
Estimated 
calories  520 


grams 

"Glidin"  34 
Sugar  20 
Lard  30 
Agar  3 
Bone  Ash  7 
Water  200 
Estimated 
calories  470 


grams 

"Glidin"  34 
Sugar  20 
Lard  30 
Agar  3 
Bone  Ash  7 
Water  200 
Estimated 
calories  470 


grama 

Meat  150 
Sugar  20 
Lard  20 
Agar  3 
Bone  Ash  7 
Water  100 
Estimated 
calories  520 


Daily  Averages 


Daily  Averages 


Daily  Averages 


Daily  Averages 


3.80 
4.16 
4.90 
+0.74 

14.5 
0.36 
2.48 

92.7 


4.63 
4.95 
4.93 
-0.02 

15.5 
0.32 
2.03 

93.6 


4.85 
5.20 
4.93 
-0.27 

15.4 
0.35 
2.25 

93.0 


3.71 
4.11 
4.93 
+0.82 

15.5 
0.40 
2.60 

91.8 


whereupon  the  meat1  and  water  were  added,2  the  whole  being 
thoroughly  mixed.  In  the  "glidin"  periods,  the  food  mixture  was 
warmed  on  the  water  bath  the  day  before  feeding,  the  water  thor- 


I 


1  Preserved  frozen,  according  to  the  method  of  Gies. 
1  Just  before  feeding. 


Utilization  of  Wheat  Proteins 


oughly  incorporated,  and  the  whole  allowed  to  stand  over  night, 
thus  giving  ample  time  for  "  hydration"1  of  the  material. 

Such  food  mixtures  were  fed  for  periods  of  3  to  5  days  to  three 
small  bitches.  The  food  was  disposed  of  in  one  meal  at  9 :00  to 
9:45  each  morning. 

TABLE  2. 

"Glidin"  with  Agar  and  Bone  Ash. 


PERIOD  IV 

(5  days) 
Meat  Feeding 

PERIOD  V 

(5  days) 
"Glidin"  Feeding 

PERIOD  VI 

(4  days) 
Meat  Feeding 

grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  3 
Bone  Ash.  7 
Water  100 
Estimated 
calories  520 

arams 

11  Glidin"  34 
Sugar  20 
Lard  30 
Agar  3 
Bone  Ash  7 
Water  200 
Estimated 
calories  470 

grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  3 
Bone  Ash  7 
Water  100 
Estimated 
calories  520 

Daily  Averages 

Daily  Averages 

Daily  Averages 

3.70 
4.06 
4.93 
+0.87 

4.83 
5.14 
4.93 
-0.21 

4.13 

4.48 
4.93 
+0.45 

13.6 
0.36 
2.64 

13.6 
0.31 
2.31 

14.5 
0.35 
2.42 

92.7 

93.6 

92.9 

SUBJECT,  DOG  6 

Weight  at  beginning,  5.0  Kg. 
Weight  at  end,  4.9  Kg. 


Composition  of  daily  diet. 


Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen,  utilization,  per 
cent  


From  an  examination  of  the  tables,  it  will  be  observed  that  the 
data  without  exception  point  to  the  fact  that  11  glidin17  is  as 
thoroughly  utilized  as  meat  under  identical  conditions. 

1  When  first  added  to  the  food  mixture  there  appeared  to  be  little  tend- 
ency for  the  water  to  be  absorbed.  On  standing  a  few  hours,  however, 
and  especially  the  next  morning,  a  thick  thoroughly  hydrated  mush  resulted. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  313 

TABLE  3. 


"Glidin"  with  Agar  and  Bone  Ash. 


SUBJECT,  DOQ  7 

Weight,  4.9  Kg. 

PERIOD  III 

(S  days) 
Meat  Feeding 

PERIOD  IV 

(5  days) 
"Glidin"  Feeding 

PERIOD  V 

(3  days) 
Meat  Feeding 

Composition  of]daily  diet.  < 

Nitrogen  output. 
Urine  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen    utilization,  per 
cent  

grams 

Meat  100 
Sugar  20 
Lard  25 
Agar  3 
Bone  Ash  7 
Water  100 
Estimated 
calories  480 

grams 

"Glidin"  23 
Sugar  20 
Lard  30 
Agar  3 
Bone  Ash  7 
Water  175 
Estimated 
calories  430 

grams 

Meat  100 
Sugar  20 
Lard  20 
Agar  3 
Bone  Ash  7 
Water  100 
Estimated 
calories  430 

Daily  Averages 

Daily  Averages 

Daily  Averages 

2.58 
2.81 
3.29 
+0.48 

12.8 
0.23 
1.81 

93.0 

3.21 
3.48 
3.29 
-0.19 

11.0 
0.27 
2.43 

91.9 

2.63 
2.89 
3.29 
+0.40 

12.7 
0.26 
2.04 

92.1 

Commercial  Gluten — Tables  4  to  9.  In  Tables  4  to  6  are 
recorded  experiments  wherein  the  utilization  of  gluten,  fed  with 
agar  and  bone  ash,  is  compared  with  meat  diets  in  which  identical 
additions  of  these  indigestible  materials  were  made.  Data  on  the 
comparative  utilization  of  gluten  and  meat,  where  no  agar  or  bone 
ash  was  employed,  are  reported  in  Tables  7  to  9. 

An  examination  of  these  tables  will  readily  convince  one  that 
the  present  sample  of  commercial  gluten  is  as  thoroughly  utilized 
as  meat. 

One  might  take  exception  to  this  generalization,  observing  that 
the  persistently  high  nitrogen  content  of  the  gluten-feces,  as  com- 
pared with  that  of  the  corresponding  meat-feces,  indicates  that  a 
portion  of  the  gluten  had  been  lost  through  the  feces.  Moreover, 


3U 


Utilization  of  Wheat  Proteins 


careful  scrutiny  will  disclose  the  fact  that  the  utilization  of  the 
gluten  is  consistently — if  only  slightly — lower  than  that  of 
meat.  This  criticism  is  indeed  valid.  However,  had  the  gluten 
been  finely  divided,  objections  of  the  above  nature,  which  in  any 
case  are  concerned  with  small  differences,  would  without  doubt  be 
untenable. 

TABLE  4. 


Gluten  with  Agar  and  Bone  Ash. 


SUBJECT,  DOG  5 

Weight  at  beginning,  5.2  Kg.   Weight  at  end,  5.2  Kg. 

PERIOD  VIII 

(5  days) 
Meat  Feeding 

PERIOD  IX* 

(6  days) 
Gluten  Feeding 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  *gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  3 
Bone  Ash  7 
Water  100 
Estimated 
calories  520 

grams 

Gluten  36 
Sugar  20 
Lard  30 
Agar  3 
Bone  Ash  7 
Water  200 
Estimated 
calories  480 

Daily  Averages 

Daily  Averages 

4.09 
4.44 
4.80 
+0.36 

15.0 
0.35 
2.32 

92.8 

4.78 
5.22 
4.90 
-0.32 

14.5 
0.44 
3.05 

91.0 

*  On  last  two  days  of  period,  about  half  the  food  was  forced. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  315 


TABLE  5. 
Gluten  with  A g ar  and  Bone  Ash. 


SUBJECT,  DOG  6 

Weight  at  beginning,  4.7  Kg.  Weight  at  end,  4.5  Kg. 

PERIOD  VIII 

(5  days) 
Meat  Feeding 

PERIOD  IX 

(5  days) 
Gluten  Feeding 

Nitrogen  output. 

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

grams 
A/Too  +              1  KCi 

Sugar  20 
Lard  20 
Agar  3 
Bone  Ash  7 
Water  100 
Estimated 
calories  520 

grams 

^jriuien  oo 
Sugar  20 
Lard  30 
Agar  3 
Bone  Ash  7 
Water  200 
Estimated 
calories  480 

Daily  Averages 

Daily  Averages 

4.23 
4.62 
4.80 
+0.18 

15.0 
0.39 
2.59 

91.9 

4.81 

5.24 
4.90 
-0.34 

14.4 

0.43 
3.00 
91.2 

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

TABLE  6. 
Gluten  with  Agar  and  Bone  Ash. 

SUBJECT,  DOG  7 

Weight  at  beginning,  4.6  Kg.   Weight  at  end,  4.6  Kg. 

PERIOD  VII 

(5  days) 
Meat  Feeding 

PERIOD  VIII 

(5  days) 
Gluten  Feeding 

Nitrogen  output. 
Urine  nitrogen,  gm  

Feces. 

Nitrogen,  gm  

grams 

Meat  100 
Sugar  20 
Lard  20 
Agar  3 
Bone  Ash  7 
Water  100 
Estimated 
calories  430 

grams 

Gluten  24 
Sugar  20 
Lard  30 
Agar  3 
Bone  Ash  7 
Water  175 
Estimated 
calories  430 

Daily  Averages 

Daily  Averages 

2.55 
2.79 
3.20 
+0.41 

12.8 
0.24 
1.86 

92.6 

3.24 
3.52 
3.27 
-0.25 

12.2 
0.28 
2.31 

91.4 

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

3i 6  Utilization  of  Wheat  Proteins 


TABLE  7. 
Gluten  without  Agar  or  Bone  Ash. 


SUBJECT,  DOG  5 

Weight  at  beginning,  5.9  Kg.    Weight  at  end,  5.8  Kg. 

PERIOD  XX 

(4  days) 
Meat  f  eeding 

PERIOD  XXII* 

(5  days) 
Gluten  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 

Feces. 

grams 

Meat  150 
Sugar  25 
Lard  20 
Water  100 
Estimated 
calories  530 

grams 

Gluten  34 
Sugar  25 
Lard  25 
Water  225 
Estimated 
calories  440 

Daily  Averages 

Daily  Averages 

4.08 
4.30 
4.59 
+0.29 

4.5 
0.22 
4.95 
95.2 

4.83 
5.05 
4.77 
-0.28 

2.8 
0.22 
7.68 
95.5 

Nitrogen,  per  cent  

*  About  half  of  the  food  forced  each  day. 

TABLE  8. 

Gluten,  without  Agar  or  Bone  Ash. 

SUBJECT,  DOG  6 

Weight  at  beginning,  6.1  Kg.    Weight  at  end,  6.2  Kg. 

PERIOD  XXI 

(4  days) 
Meat  Feeding 

PERIOD  XXIII 

(5  days) 
Gluten  Feeding 

Composition  of  daily  diet   < 

Nitrogen  output. 

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

grams 

Meat,  150 
Sugar  25 
Lard  20 
Water  100 
Estimated 
calories  530 

grams 

Gluten  34 
Sugar  25 
Lard  25 
Water  225 
Estimated 
calories  440 

Daily  Averages 

Daily  Averages 

3.55 
3.77 
4.59 
+0.82 

3.5 
0.22 
6.34 
95.2 

4.92 
5.17 
4.77 
-0.40 

3.6 
0.25 
6.93 
94.8 

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

Lafayette  B.  Mendel  and  Morris  S.  Fine  317 

TABLE  9. 


Gluten  without  Agar  or  Bone  Ash. 


SUBJECT,  DOG  7 

Weight  at  beginning,  5.9  Kg.   Weight  at  end,  6.3  Kg. 

PERIOD  XX 

(4  days) 
Meat  Feeding 

PERIOD  XXII 

(5  days) 
Gluten  Feeding 

Composition  of  daily  diet  I 

Nitrogen  output. 
Total  nitrogen,  gm  

Feces. 

Weight  air  dry,  gm  

grams 

Meat  150 
Sugar  25 
Lard  20 
Water  100 
Estimated 
calories  530 

grams 

Gluten  34 
Sugar  25 
Lard  25 
Water  225 
Estimated 
calories  440 

Daily  Averages 

Daily  Averages 

3.55 
3.74 
4.59 
+0.85 

3.2 
0.19 
5.93 
95.8 

4.68 
4.81 
4.77 
-0.04 

1.8 
0.13 
7.40 
97.2 

Glutenin — Tables  10,  11.  Man,  Table  10:  The  subject  of  this 
experiment,  M.  S.  F.,  was  23  years  of  age,  about  57.5  kilos  in  weight. 
Throughout  the  experiment  he  was  engaged  in  the  routine  con- 
nected with  such  work.  The  general  plan  of  the  experiment  was 
as  follows :  A  preliminary  period  of  three  days  was  obtained,  dur- 
ing which  the  body  was  enabled  to  attain  nitrogen  equilibrium  and 
to  adjust  itself  to  the  experimental  conditions.  As  will  be  noted 
from  the  accompanying  table,  during  the  preliminary,  fore,  and 
after  periods,  a  varied  mixed  diet  was  consumed;  and  during  the 
experimental  period,  the  meat,  nut  butter  and  part  of  the  egg  were 
replaced  by  glutenin. 


3i8 


Utilization  of  Wheat  Proteins 


Character  of  the  Diet. 


PRELIMINARY,  FORE 
AND  AFTER  PERIODS 

EXPERIMENTAL  PERIOD 

Daily  Averages 

Daily  Averages 

Gm. 

Gm. 

Cereal  

20 

20 

60 

60 

Egg  

110 

50 

Pine  nut  butter  

50 

Meat  

110 

62 

Potato  

110 

110 

Banana  

140 

140 

270 

250 

160 

180 

Milk  

40 

40 

Sugar  

110 

180 

Butter  

40 

80 

Cereal  coffee,  tea  

600 

600 

TABLE  10. 
Glutenin. 


SUBJECT,  MAN 

Weight  at  beginning,  57.8  Kg. 
Weight  at  end,  57.6  Kg. 


Composition  of  daily  diet. 


Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm — 
Nitrogen  balance,  gm.... 
Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per 
cent  


PERIOD  I 

(6  days) 
Mixed  Diet 


Meat 
nut  butter, 
cereal,potato 
fruit,  etc. 


Estimated 
calories  2400 


Daily  Averages 


9.85 
11.10 
11.30 
+0.25 

23.0 
1.25 
5.45 

89.0 


PERIOD  II 

(4  days) 
Glutenin 


Glutenin 
cereal,  potato, 
fruit,  etc. 

67.2  per  cent 
of  the  total 
nitrogen  fur- 
nished by 
glutenin. 

Estimated 
calories  2500 


Daily  Averages 


10.80 
12.11 
11.12 
-0.99 

22.1 
1.31 

5.92 

88.2 


PERIOD  III 

(4  days) 
Mixed  Diet 


Meat,  eggs, 
nut  butter, 
cereal, potato, 
fruit,  etc. 


Estimated 
calories  2600 


Daily  Averages 


9.98 
11.36 
11.15 
-0.21 

24.7 
1.38 
5.63 

87.6 


Lafayette  B.  Mendel  and  Morris  S.  Fine  319 


These  diets  furnished  about  11.2  grams  nitrogen  and  2,500 
calories  per  day;  during  the  experimental  period  67  per  cent  of 
the  nitrogen  was  supplied  by  the  glutenin. 

During  the  evening  of  the  third  day  of  the  experimental  period, 
slight  indigestion  was  manifest,  which  continued  to  the  following 
morning,  when  100  cc.  of  0.2  per  cent  HC1  were  taken.1  The  glu- 
tenin had  a  peculiar  ether-alcohol  odor  which  could  not  be  removed 
by  drying  at  130°  C.  for  eight  consecutive  hours.  This  odor  was 
very  disagreeable  and  nauseating  and  could  not  be  covered  by 
mixing  with  other  foods.  It  was  found  that  mixing  the  glutenin 
with  egg  and  potato,  and  frying  this  mixture,  was  practically  the 
only  way  in  which  the  glutenin  could  be  consumed  in  any  quantity. 
Under  such  circumstances  it  is  probable  that  the  psychic  secretion 
was  a  negligible  quantity  and  this  may  account  for  the  symptoms 
of  indigestion  noted. 

With  the  exception  of  the  feeling  of  nausea  attending  the  meals 
of  the  four  days  of  glutenin  feeding,  the  subject  felt  perfectly  well. 
Throughout  the  experiment  defecation  was  accomplished  regu- 
larly every  morning,  the  volume  of  feces  being  apparently  alike 
from  day  to  day.  The  feces  were  of  semi-solid  consistency,  never 
well  formed.  There  was,  however,  nothing  approaching  diarrhoea, 
throughout  the  experiment,  except  on  the  last  day  of  glutenin 
feeding. 

In  spite  of  the  nausea  with  its  possible  secretory  consequences, 
the  glutenin  appeared  to  be  as  well  digested  as  the  materials  which  it 
replaced  in  the  control  diets.  It  should  be  observed,  however,  that 
about  25  per  cent  of  the  nitrogen  of  the  control  diets  was  furnished 
by  nut  butter,  and,  according  to  Jaffa,2  the  nitrogen  of  nuts  is  in 
general  only  about  75  per  cent  utilized.  Hence,  in  order  to  show 
the  readily  digestible  nature  of  glutenin,  the  coefficient  of  diges- 
tibility in  the  experimental  period  should  have  been  somewhat 
greater  than  those  for  the  control  periods.  Nevertheless,  it  is 
significant  to  note  that  in  two  experiments  on  the  same  subject, 
where  mixed  diets  were  employed,  which  contained  no  nut  prep- 
arations but  were  otherwise  similar,  the  utilization  was  88  and 
86  per  cent,  respectively. 

1  This  affects  one  day  out  of  four;  and  since  the  HC1  gave  no  noticeable 
relief,  the  influence  of  the  acid  even  in  this  day  was  probably  very  small. 
*  Jaffa:    Office  of  Experiment  Stations,  Bull.  132,  p.  69,  1903. 


320 


Utilization  of  Wheat  Proteins 

TABLE  11. 

Glutenin  with  Agar  and  Nutrient  "Salts." 


SUBJECT,  DOG  4 

Weight  at  beginning,  5.1  Kg. 
Weight  at  end,  5.0  Kg. 


Composition  of  daily  diet . 


Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per 
cent  


PERIOD  I 

(5  days) 
Meat  Feeding 

PERIOD  II* 

(5  days) 
Glutenin  Feeding 

PERIOD  III 

(3  days) 
Meat  Feeding 

grams 

Meat  150 

grams 

Glutenin  43 

grams 

Meat  150 

Sugar  15 
Starch  5 

Sugar  25 
Starch  5 

Sugar  25 
Starch  5 

Lard  20 

Lard  30 

Lard  20 

Agar  8 
Salts  2 

Agar  8 
Salts  4 

Agar  8 
Salts  4 

Water  200 

tit    j  _  nr\r\ 

Water  300 

Water  200 

Estimated 

Estimated 

Estimated 

calories  520 

calories  520 

calories  560 

Daily  Averages 

Daily  Averages 

Daily  Averages 

4.71 

4.71 

4.18 

5.04 

5.01 

4.48 

5.18 

5.18 

5.18 

+0.14 

+0.17 

+0.70 

13.6 

15.0 

12.7 

0.33 

0.30 

0.31 

2.40 

1.99 

2.42 

93.7 

94.3 

94.1 

*  About  half  of  the  food  forced  each  day. 


For  mixed  diets  on  man  93  per  cent  is  given  as  the  average  coef- 
ficient of  digestibility  by  At  water  and  Bryant,1  being  considerably 
higher  than  the  coefficients  obtained  in  our  experiments.  The 
difference  may  be  accounted  for  by  the  presence  in  the  diets  of 
relatively  large  amounts  of  fruits  and  vegetables,  which  contain 
considerable  indigestible  material.2 

Dog,  Table  11:  The  plan  of  the  experiment  did  not  differ  essen- 
tially from  those  already  described.    The  character  of  the  indi- 

1  See  footnote,  1  p.  304. 

2  Cf.  Bryant  and  Milner :    American  Journal  of  Physiology,  x,  p.  96,  1903. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  321 


gestible  materials  was  somewhat  different,  larger  amounts  of  agar 
being  used,  without  bone  ash,  the  latter  being  replaced  by  a  salt 
mixture.1  Despite  the  necessity  of  forced  feeding,  the  glutenin 
was  quite  as  thoroughly  utilized  as  meat  fed  under  similar  experi- 
mental conditions. 

Gliadin— Tables  12,  13.  Man,  Table  12:  The  plan  of  the 
experiment  was  essentially  that  outlined  under  glutenin,  except 
that  slightly  more  meat  was  eaten  in  the  control  periods;  and  in 
the  gliadin  period  the  egg,  meat  and  nut  butter  were  completely 
replaced  by  gliadin,  which  supplied  85  per  cent  of  the  total  nitro- 
gen. The  gliadin  when  mixed  with  water  forms  a  veritable  glue, 
which  cannot  possibly  be  eaten.  This  glue-like  consistency  was 
avoided  by  mixing  with  cornstarch,  salt,  sugar,  and  baking  powder, 
which  mixture  was  finally  baked.  Even  in  this  condition,  the 
material  soon  evoked  nausea  which,  on  the  last  day  of  the  period, 
made  a  discontinuance  necessary.  Aside  from  the  nausea  attend- 
ing the  meals  of  the  gliadin  period,  the  subject  felt  in  excellent  con- 
dition throughout  the  experiment,  diarrhoea  and  symptoms  of 
indigestion  being  entirely  absent. 

From  Table  12,  it  is  evident  that  gliadin  is  as  thoroughly  utilized 
as  the  materials  of  the  control  periods,  which  it  replaced. 

The  salts  of  the  baking  powder  seemed  to  have  no  appreciable 
influence.  The  apparently  low  digestibility  of  the  mixed  diets 
has  already  been  discussed  under  glutenin. 

Dog,  Table  IS:  The  plan  of  this  experiment  was  exactly  the 
same  as  that  described  on  page  320  under  the  glutenin  experiment 
on  a  dog.    The  food  mixture  was  like  so  much  glue,  but  the  animal 

1  With  one  or  two  modifications  this  is  the  salt  mixture  proposed  by 
Rohmann  (Allgemeine  medizinische  Central-Zeitung,  No.  9,  1908).  Itcon. 
sisted  of  the  following  ingredients: 

Gram 


Calcium  phosphate   10 

Acid  potassium  phosphate   37 

Sodium  chloride   20 

Sodium  citrate   15 

Magnesium  citrate   8 

Calcium  lactate   8 

Ferric  citrate   2 


322 


Utilization  of  Wheat  Proteins 


TABLE  12. 

It/?  nni  Yl 

SUBJECT,  MAN 

PERIOD  I 

PERIOD  II 

PERIOD  III 

Weight  at  beginning,  57.0  Kg. 

(5  days) 

(4  days) 

(4  days) 

Weight  at  end,  57.6  Kg. 

Mixed  Diet 

Gliadin 

Mixed  Diet 

ivleat,  eggs, 

tjriiauin, 

Meat,  eggs, 

nut  butter, 

potatoes, 

nut  butter, 

potatoes, 

fruit,  etc. 

potatoes, 

fruit,  etc. 

84.8  percent  of 

fruit,  etc. 

Composition  of  daily  diet.  < 

the  total  ni- 

trogen sup- 

plied by  glia- 

din. 

Estimated 

Estimated 

Estimated 

calories  2600 

calories  2800 

calories  2900 

Daily  Averages 

Daily  Averages 

Daily  Averages 

iV itrogen  output. 

in  79 

1U .  i  z 

11 .  OO 

10  in 

1U.  1U 

19  1Q 

LZ .  lo 

1Z  .00 

1 1  QQ 
11  .OO 

19  79 
LZ .  i  Z 

LZ .  \JZ 

19  ftfi 
1Z  .  Do 

Nitrogen  balance,  gm  

+0.60 

-0.80 

+1.30 

Feces. 

Weight  air  dry,  gm  

23.6 

22.5 

26.8 

Nitrogen,  gm  

1.41 

1.27 

1.53 

Nitrogen,  per  cent  

5.96 

5.53 

5.74 

Nitrogen  utilization,  per 

cent  

89.0 

89.4 

87.9 

ate  it  with  apparent  relish.  From  Table  13  the  glutenin  appears 
to  have  been  quite  thoroughly  utilized. 

Abderhalden  obtained  plus  balances  with  gliadin,  but  notes 
that  his  preparations  contained  0.35  per  cent  lysine — -an  amino- 
acid  not  found  in  pure  gliadin.  He  infers  further  that  it  is  not 
known  whether  really  pure  gliadin  can  maintain  nitrogen  equilib- 
rium. We  are  unable  to  state  whether  or  not  the  gliadin  employed 
in  our  experiments  was  free  from  lysine,  but  the  negative  nitrogen 
balances,  even  with  large  intakes,  are  significant.  Henriques  ob- 
tained plus  balances  with  gliadin  in  rats,  but  Abderhalden  ques- 
tions the  purity  of  his  preparations.1 

1  Osborne  and  Mendel  (Feeding  Experiments  with  Isolated  Food-Sub- 
stances, Carnegie  Institution  of  Washington,  Publication  No.  156.  p.  21, 


Lafayette  B.  Mendel  and  Morris  S.  Fine  323 

TABLE  13. 


Gliadin  with  Agar  and  Nutrient  "Salts." 


SUBJECT,   DOG  4 

PERIOD  III 

PERIOD  IV 

PERIOD  V 

Weight  at  beginning,  4.9  Kg. 

(3  days) 

(5  days) 

(4  days) 

Weight  at  end,  4.9  Kg. 

Meat  Feeding 

Gliadin  Feeding 

Meat  Feeding 

gram 

gram 

gram 

Meat  150 

Gliadin  32 

Meat  150 

ougar  60 

ougar  ZiO 

ougar  ao 

Starch  5 

Starch  5 

Starch  5 

Lard  20 

Lard  30 

Lard  20 

Oomnosition  of  dailv  dipt  < 

Agar  8 

Agar  8 

Bone  Ash  10 

Salts  4 

Salts  4- 

Water  200 

Water  200 

Water  250 

TT!g f"  1  TY\  Q  "f  ori 

TTlcf"!  m  {jfon 

TT.stim  n  tpd 

cfiloriGS  520 

calories  560 

Daily  Averages 

Daily  Averages 

Daily  Averages 

Nitrogen  output. 

Urine  nitrogen,  gm  

4.18 

5.09 

4.24 

Total  nitrogen,  gm  

4.48 

5.37 

4.50 

Nitrogen  in  food,  gm  

5.18 

5.10 

5.40 

Nitrogen  balance,  gm  

+0.70 

-0.27 

+0.90 

Feces . 

12.7 

13.4 

15.0 

Nitrogen,  gm  

0.31 

0.28 

0.27 

Nitrogen,  per  cent  

2.42 

2.07 

1.79 

Nitrogen  utilization,  per 

cent  

94.1 

94.5 

95.0 

It  is  difficult  at  present  to  account  for  the  persistent  negative 
balances  with  the  other  protein  preparations.  Michaud  did  indeed 
obtain  minus  balances  with  "  gliding '  but  his  nitrogen  intakes  were 
very  small,  while  those  in  our  experiments  were  relatively  large. 


1911)  state  that  in  rat  experiments  at  least  10  per  cent  of  the  excreted  nitro- 
gen may  be  lost  in  connection  with  the  difficult  manipulation  attending 
metabolism  experiments  with  small  animals.  This  condition  may  in  part 
account  for  the  positive  balances  obtained  by  Henriques. 


324  Utilization  of  Wheat  Proteins 

Nitrogen  Balances. 

TABLE  14. 


Average  Daily  Nitrogen  Balances  for  the  Wheat  Proteins  and  Correspond- 
ing Values  for  Meat. 


SUBJECT 

TABLE 

"glidin" 

"gluten" 

GLUTENIN 

GLIADIN 

MEAT 

Dog  5.. 

i 

-0.02,-0.27 



+0.74,-1-0.82 

Doer 

£i 

—  0  91 

in  07   in  &K 

Dog  7.. 

3 

-0.19 

+0.48, +0.40 

Dog  5.. 

4 

-0.32 

+0.36 

Dog  6. . 

5 

-0.34 

+0.18 

Dog  7.. 

6 

-0.25 

+0.41 

Dog  5.. 

7 

-0.28 

+0.29 

Dog  6.. 

8 

-0.40 

+0.82 

Dog  7.. 

9 

-0.04 

+0.85 

Man  . . . 

10 

-0.99 

+0.25,-0.21 

Dog  4. . 

11 

+0.17 

+0.14, +0.70 

Man  . . . 

12 

-0.80 

+0.60,+1.30 

Dog  4. . 

13 

-0.27 

+0.70,  +0.90 

SUMMARY. 

The  problems  associated  with  the  utilization  of  food  products 
of  plant  origin  have  been  reviewed  as  an  introduction  to  a  series 
of  experimental  studies  on  the  nutritive  value  of  vegetable  pro- 
teins. It  is  pointed  out  that  two  distinct  questions  must  be  con- 
sidered, namely:  (1)  the  availability  of  the  products  existing  more 
or  less  in  their  native  condition,  with  accompanying  structural 
elements,  as  in  bread;  (2)  the  specific  utilization  of  the  proteins 
themselves.  The  latter  aspect  is  the  one  which  primarily  calls 
for  further  investigation. 

In  our  feeding  experiments  an  attempt  has  been  made  to  con- 
trol the  extraneous  factors  as  far  as  possible,  by  improving  the 
texture  and  mechanical  condition  of  the  crude  products,  or  puri- 
fying the  individual  proteins.  The  present  paper  deals  with  wheat. 
The  experimental  trials  on  man  and  dogs  indicate  that  "glidin," 
gluten,  and  the  two  characteristic  proteins  of  wheat,  gliadin  and  glu- 
tenin,  are  as  thoroughly  utilized  as  the  nitrogenous  components  of 
fresh  meat. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  325 


BIBLIOGRAPHY. 

Abderhalden:    1909,  Zeitschrift  fiir  physiologische  Chemie,  Ix,  p.  418. 

Bischoff,  E. :    1869,  Zeitschrift  fur  Biologie,  v,  p.  454. 

Bischoff,  T.  and  Voit:    "Die  Erndhrung  des  Fleischfressers." 

Bornstein:    1897,  Berliner  klinische  Wochenscrhift,  No.  8,  p.  162. 

Buslik  and  Goldhaber:  1911,  Zeitschrift  fiir  physikalische  und 
didtetische  Therapie,  xv,  p.  93. 

Constantinidi:    1887,  Zeitschrift  fiir  Biologie,  xxiii,  p.  433. 

Erismann:    1901,  Zeitschrift  fiir  Biologie,  xlii,  p.  672. 

Fauvel:  1907,  Maly's  Jahresbericht  iiber  die  Fortschritte  der  Tierchemie, 
xxxvii,  p.  605. 

Henriques:    1909,  Zeitschrift  fiir  physiologische  Chemie,  lx,  p.  105. 
Kornauth:    1892,  Oesterreichisches  landwirtschaftliches  Centralblatt. 
Laves:    1900,  Miinchener  medizinische  Wochenschrift,  p.  1339. 
Lehmann,  K.  B. :    1894,  Archiv  fiir  Hygiene,  xx,  p.  1;  1902,  ibid.,  xlv, 
p.  177. 

Lusk:    1890,  Zeitschrift  fiir  Biologie,  xxvii,  p.  459. 

Menicanti  and  Prausnitz  :    1894,  Zeitschrift  fur  Biologie,  xxx,  p.  328. 

Meyer,  G.:    1871,  Zeitschrift  fiir  Biologie,  vii,  p.  1. 

Michaud  :    1909,  Zeitschrift  fiir  physiologische  Chemie,  lix,  p.  405. 

Potthast:    1887,  Inaugural  Dissertation,  Leipzig 

Prausnitz  :    1893,  Archiv  fiir  Hygiene,  xvii,  p.  626. 

Romberg:    1897,  Archiv  fiir  Hygiene,  xxviii,  p.  274. 

Rubner:    1879,  Zeitschrift  fiir  Biologie,  xv,  p.  115;  1883,  ibid.,  xix,  p.  45. 

Salkowski:    1909,  Bioche.vAsche  Zeitschrift,  xix,  p.  83. 

Wicke:  1890.  Archiv  fiir  Hygiene,  xi,  p.  349  (see  the  corrections  made 
by  Rubner,  ibid.,  xiii,  p.  123,  1891). 

Wintgen:  1902,  Zeitschrift  fiir  Untersuchung  der  Nahrungs-  und  Ge- 
nussmittel,  v,  p.  289. 

Woods  and  Merrill:    1900,  Office  of  Experiment  Stations,  Bull.  85. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  X,  No.  4.  1911 


STUDIES  IN  NUTRITION. 
H.    THE  UTILIZATION  OF  THE  PROTEINS  OF  BARLEY. 

By  LAFAYETTE  B.  MENDEL  and  MORRIS  S.  FINE. 

(From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  September  25,  1911.) 

Data  regarding  the  utilization  of  the  proteins  of  barley  are  con- 
fined almost  entirely  to  the  Japanese  literature,1  according  to 
which  they  are  but  40  to  76  per  cent  available.  However,  the 
experiments  from  which  these  data  are  taken  were  not  exempt 
from  those  unfavorable  conditions  which  render  it  difficult  to 
draw  conclusions.2 

METHODS. 

The  routine  incident  to  the  metabolism  experiments  did  not 
differ  essentially  from  that  described  in  the  previous  paper  of 
this  series.  The  concentration  of  hemicelluloses  in  the  barley 
preparation  and  in  the  resulting  feces  was  determined.  For  this 
purpose  2.5  to  3.5  grams  of  the  dried  material  were  boiled  for  four 
hours  with  220  cc.  of  2  per  cent  hydrochloric  acid.3  Proteoses  and 
peptones,  which  disturb  the  subsequent  precipitation  of  copper 
oxide,  were  removed  by  the  addition  of  a  solution  of  phosphotung- 

1  Cf .  Oshima:  U.  S.  Department  of  Agriculture,  Office  of  Experiment 
Stations,  Bull.  159,  1905. 

2  Cf.  Mendel  and  Fine:    This  Journal,  x,  p.  303,  1911. 

3  From  the  studies  of  Swartz  (Transactions  of  the  Connecticut  Academy 
of  Arts  and  Sciences,  xvi,  p.  323,  1911)  it  appears  that  maximum  reduction 
after  hydrolysis  is  obtained  in  three  to  four  hours.  The  crude  barley  pro- 
tein preparation  of  these  experiments  yielded  but  0.6  per  cent  more  hemi- 
celluloses when  boiled  four  hours  than  when  the  interval  was  reduced  to  two 
and  a  half  hours  as  called  for  in  the  official  method  for  the  estimation  of 
starch  (U.  S.  Dept.  of  Agriculture,  Bureau  of  Chemistry,  Bull.  107,  p.  53, 
1910). 

339 


340 


Utilization  of  Barley  Proteins 


stic  acid.1  The  resulting  precipitate  was  washed,  the  united  fil- 
trates neutralized  and  made  up  to  500  cc,  of  which  25  cc.  were 
used  for  the  sugar  estimation  according  to  the  Allihn  gravimetric 
method.  The  hemicellulose  was  computed  by  multiplying  the 
dextrose  thus  obtained  by  O.9.2 

EXPERIMENTAL  PART. 

Product  Employed. 

The  crude  barley  protein  employed  in  this  study  was  prepared 
as  follows: 

About  seven  pounds  of  barley  flour,3  in  one  pound  lots,  were  made  into 
a  thin  homogeneous  mush  with  water  and  heated  in  an  autoclave  at  about 
120°  C.  for  ten  to  twenty  minutes.  After  the  material  had  cooled  to  a  tem- 
perature of  about  60°  C.  an  amylolytic  preparation  was  added.  It  was  neces- 
sary to  repeat  this  process  at  least  three  times  before  the  cells  were  com- 
pletely disintegrated  and  no  starch  reaction  was  obtained  in  a  test  tube 
trial.  The  insoluble  material  thus  obtained  settled  very  readily  and  was 
washed  with  ease  four  to  six  times  with  water  by  decantation.  It  was 
finally  filtered,  dried  on  a  water  bath  and  ground  to  an  impalpable  powder. 
Analysis  gave  the  following  values: 

per  cent 


Protein  (N  X  5.74)   51.0 

Carbohydrate  by  hydrolysis  (hemicelluloses)   21.7 

Ether  extract   8.3 

Ash   1.0 

Moisture   3.0 

Crude  fiber  (by  difference).    15.0 


When  treated  with  iodine  and  examined  under  the  microscope, 
starch  particles  were  so  infrequent  as  to  be  considered  absent;  yet 

1  Cf.  Abderhalden' s  Handbuch  der  Biochemischen  Arbeitsmethoden,  iii, 
1,  p.  271,  1910.  With  this  method  the  barley  preparation  yielded  21.7  per 
cent  hemicelluloses;  when  the  phosphotungstic  precipitation  was  omitted, 
the  yield  was  reduced  to  18.5  per  cent.  Swartz  (loc.  cit.,  p.  343)  adopted  the 
expedient  of  clarifying  with  charcoal  with  satisfactory  results. 

2  This  is  believed  to  be  justifiable  in  view  of  the  similarity  in  ultimate 
analysis  of  starch  and  hemicelluloses. 

3  Mr.  M.  F.  Deming  of  the  Cereo  Company,  Tappan,  N.  Y., kindly  con- 
tributed this  material. 

4  Factor  proposed  by  Atwater  and  Bryant :  Report  of  the  Storrs  Agri- 
cultural Experiment  Station,  p.  79,  1899. 


Lafayette  B.  Mendel  and  Morris  S.  Fine      34 j 


on  hydrolyzing  with  dilute  acid,  22  per  cent  of  carbohydrate  was 
obtained.  This  condition  would  suggest  that  the  values  for  starch, 
as  ordinarily  found  by  acid  hydrolysis,  cannot  be  relied  upon, 
since  a  body  other  than  starch  may  be  present,  which  on  hydroly- 
sis yields  reducing  substances.  Indeed  Schulze1  has  shown  the 
wide-spread  occurrence  of  hemicelluloses,  a  group  of  substances 
readily  attacked  by  dilute  acids,  yielding  reducing  sugars,  and  only 
very  slowly  affected  by  enzymes.2  Apparently  the  untreated 
barley  contains  at  least  5  per  cent  of  hemicelluloses. 

Metabolism  Experiments. 

Crude  barley  protein  was  fed  to  two  bitches  as  detailed  in  the 
first  two  tables.  Casual  inspection  of  these  data  would  indicate 
that  barley  protein  is  relatively  poorly  utilized,  having  a  coeffi- 
cient of  digestibility  of  85  per  cent  against  97  per  cent  for  meat 
fed,  as  it  might  at  first  sight  appear,  under  identical  conditions. 
However,  each  day's  supply  of  barley  protein  preparation  contained 
about  8  grams  of  crude  fiber — practically  indigestible3 — and  11 
grams  of  hemicelluloses,  of  which  9  grams  reappeared  in  the  feces 
in  both  experiments.4  The  barley-protein  feces  thus  contained 
about  17  grams  of  undigested  non-nitrogenous  material,  and  these 
experiments  should  therefore  not  be  held  comparable  to  trials 
with  meat  where  such  unfavorable  conditions  were  not  in  evi- 
dence. 

1  Cf.  Schulze  and  Godet:    Zeitschr.f.  physiol.  Chem.,  lxi,  p.  281, 1909. 

2  Cf.  Swartz:  hoc.  cit.  (contains  the  literature). 

3  Cf.  Swartz:  hoc.  cit.,  p.  268  ff.  (contains  the  literature). 

4  The  barley-protein  feces  from  both  animals  yielded  10  grams  of  dextrose 
(=  9  grams  of  hemicellulose)  on  hydrolysis.  The  criticism  might  be  offered 
that  a  portion  of  the  dextrose  thus  obtained  is  due  to  the  cane  sugar  of  the 
food  mixture,  its  digestion  and  absorption  having  been  diminished  by  the 
excessive  fecal  discharges  (22  grams  dry  daily).  Against  this  objection 
are  the  following  facts :  (1)  meat  diets  containing  10  grams  of  bone  ash  but 
otherwise  identical  to  those  in  this  paper  yielded  14  to  15  grams  of  dry  carbo- 
hydrate-free feces  daily;  (2)  cotton  seed  diets,  containing  25  grams  of  cane 
sugar  as  in  the  above  trials,  produced  23  to  24  grams  of  dry  feces,  which 
yielded  only  4  to  6  grams  of  reducing  carbohydrate  on  hydrolysis — quantities 
attributable  to  undigested  hemicelluloses  of  the  food. 


342 


Utilization  of  Barley  Proteins 


The  importance  of  comparing  experiments  in  which  the  indiges- 
tible non-nitrogenous  material,1  in  addition  to  the  nitrogen  intake 
and  accessory  articles  of  diet,  are  similar,  was  adequately  appre- 
ciated only  after  the  major  portion  of  the  studies  of  this  series  was 
completed.  Trials  in  which  these  principles  were  consistently 
applied  not  being  at  hand,  we  must  be  content  with  grouping  data 
which  will  as  far  as  possible  enable  one  to  interpret  properly  the 
results  on  protein  utilization.  Table  3  contains  such  an  arrange- 
ment. Meat  diets  containing  6  grams  of  fiber  and  13  grams  of 
indigestible  materials  were  91  and  89  per  cent  utilized,  respectively. 
Thus  the  utilization  of  85  per  cent  for  the  barley  preparation  with 
its  8  grams  of  fiber  and  11  grams  of  hemicelluloses  (of  which  9 
grams  reappeared  in  the  feces)  leads  one  to  believe  that  under 
favorable  conditions,  barley  protein,  like  that  of  the  closely  related 
cereal  wheat,  would  be  almost  perfectly  digested. 


TABLE  1. 

Crude  Barley  Protein. 


SUBJECT,  DOG  5 

PERIOD  XXVIII 

PERIOD  XXIX* 

Weight  at  beginning,  6.2  Kg.     Weight  at 

(5  days) 

(4  days) 

end,  6.3  Kg. 

Meat  Feeding 

Barley  Protein  Feeding 

grams 

grams 

Meat  150 

Barley  protein  52 

Sugar  25 

Sugar  25 

Composition  of  daily  diet  ( 

Lard  20 

Lard  30 

Water  100 

Water  200 

Estimated 

Estimated 

calories  530 

calories  490 

Daily  Averages 

Daily  Averages 

Nitrogen  output 

Urine  nitrogen,  gm  

3.68 

3.99 

Total  nitrogen,  gm  

3.84 

4.67 

Nitrogen  in  food,  gm  

4.64 

4.64 

Nitrogen  balance,  gm  

+0.80 

-0.03 

Feces 

Weight  air  dry,  gm  

3.4 

22.0 

Nitrogen,  gm  

0.16 

0.68 

Nitrogen,  per  cent  

4.62 

3.10 

Nitrogen  utilization,  per  cent  

96.6 

85.3 

•About  half  the  food  forced. 


1  The  influence  of  such  substances  upon  nitrogen  utilization  will  be  dis- 
cussed more  fully  in  a  subsequent  paper. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  343 

TABLE  2. 


Crude  Barley  Protein.. 


SUBJECT,  DOG  7 

PERIOD  XXVIII 

PKRIOD  XXtX 

Weight  at  beginning,  6.6  Kg.    Weight  at 

(5  days) 

(5  days) 

end,  6.7  Kg. 

Meat  Feeding 

Barley  Protein  Feeding 

grams 

qtcliyis 

Meat 

150 

Barley  protein  52 

Sugar 

25 

Sugar  25 

Composition  of  daily  diet   < 

Lard 

20 

Lard  30 

Water 

100 

Water  200 

Estimated 

Estimated 

calories 

530 

calories  490 

Nitrogen  output 

Daily  Averages 

Daily  Averages 

3.60 

3.75 

Total  nitrogen,  gm  

3.75 

4.45 

Nitrogen  in  food,  gm  

4.64 

4.64 

Nitrogen  balance,  gm  

+0.89 

+0.19 

Feces 

3.8 

22.4 

Nitrogen,  gm  

0.15 

0.70 

Nitrogen,  per  cent  

3.82 

3.12 

Nitrogen  utilization,  per  cent  

96.9 

85.0 

TABLE  3. 

Utilization  with  Reference  to  Indigestible  Materials  in  the  Diet.  Daily  Averages. 


DOG 

PERIOD 

DAYS 

NATURE  OF  INGESTA 

FIBER 
IN  OR 
ADDED 
TO  FOOD 

TOTAL 
INDIGES- 
TIBLE 
MATERIAL 
IN  FOOD 

N 

INTAKE 

N 

UTILIZA- 
TION 

AVER- 
AGE N 
UTILIZA- 
TION 

grams 

grams 

grams 

per  cent 

per  cent 

5 

xxix 

4 

1  Crude  barley  f 

8 

19t 

4.6 

85.3 

|  85.2 

7 

xxix 

5 

/     protein  \ 

8 

19t 

4.6 

85.0 

5 

xviii* 

4 

6 

6 

3.3 

90.5 

6 

xix* 

4 

J  Meat  | 

6 

6 

3.3 

89.2 

J  91.0 

7 

xviii* 

4 

6 

6 

3.3 

93.3 

5 

XV* 

4 

1  Meat 

6 

13 

3.3 

91.6 

6 

xvi* 

4 

\  AgarJ  2  gm.  j 

6 

13 

3.3 

87.7 

J  89.2 

7 

XV* 

4 

J  Bone  ash§  5  gm.  [ 

6 

13 

3.3 

88.3 

*  The  details  of  these  experiments  will  be  published  in  a  subsequent  paper  of  this  series, 
t  This  includes  11  grams  of  hem icelluloses,  which  were  18  per  cent  utilized  (see  page  341, 
this  paper). 

JSaiki  (this  Journal,  ii,  p.  251,  1906)  recovered  all  but  17  per  cent  of  the  agar  fed.  The 
amount  of  agar  thus  failing  to  reappear  in  the  feces  of  these  experiments  is  too  small  to  be 
considered. 

§  We  are  unable  to  state  exactly  how  completely  the  bone  ash  reappears  in  the  feces.  Steel 
and  Gies  (Amer.  Journ.  of  Physiol.,  xx,  p.  350,  1907)  found  no  change  in  urinaiy  calcium  or 
phosphorus  when  large  amounts  of  bone  ash  were  added  to  the  diet. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  X,  No.  5,  1911 


STUDIES  IN  NUTRITION. 
III.    THE  UTILIZATION  OF  THE  PROTEINS  OF  CORN. 

By  LAFAYETTE  B.  MENDEL  and  MORRIS  S.  FINE. 

(From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  September  25,  1911.) 

Corn  has  been  produced  extensively  as  a  food  for  man  in  America 
for  many  hundreds  of  years  1  It  is  scarcely  a  century  since  corn 
has  ceased  to  be  the  most  important  of  our  food  cereals,  this  high 
place  in  our  dietary  having  since  been  assumed  by  wheat  because 
of  its  superior  bread-making  qualities.  Corn  has  been,  and  is, 
an  important  article  of  diet  in  other  parts  of  the  world  also.  A 
very  popular  dish  in  Italy  is  maize  cooked  with  water  to  a  stiff 
mush,  to  which  a  little  cheese  is  added.  This  preparation  is  called 
polenta,  and.  according  to  Ranke,2  it  is  the  main  article  of  diet 
in  certain  parts  of  Italy. 

Rubner  reported  a  study  on  the  availability  of  polenta  seasoned 
with  meat  extracts  The  digestibility  of  the  whole  was  85  per 
cent,  but  assuming  the  meat  extracts  to  be  completely  absorbed, 
the  apparent  utilization  would  fall  to  80  per  cent. 

In  experiments  on  himself  Malfatti  found  the  digestibility  of 
maize  to  be  82  per  cent.  The  utilization  of  this  material  was 
lowered  to  68  per  cent  by  the  addition  of  much  butter  and  raised 
to  93  per  cent  when  cheese  was  added  to  the  diet. 

The  work  of  Grandeau,  on  the  availability  of  maize  in  the  horse, 
is  of  interest  in  that  it  shows  that  even  in  an  animal  with  a  long 
intestine,  where  the  food  may  remain  for  a  greater  length  of  time, 
and  the  cellulose  be  dissolved  to  a  considerable  extent — even  here 
the  digestibility  was  an  average  of  only  69  per  cent.  The  gener- 
ally low  utilization  of  polenta  is  further  shown  by  Albertoni  and 
Novi,  and  by  Erismann. 

1  Cf .  Merrill  (1906) :  see  bibliography. 

2Ranke:    Zeitschrift  fur  Biologie,  xiii,  p.  130,  1877. 

345 


34^ 


Utilization  of  Corn  Proteins 


The  utilization  experiments  of  Merrill,  in  which  the  protein  of 
the  corn  of  various  corn  preparations  was  found  to  vary  in  diges- 
tibility from  73  to  86  per  cent,  lend  further  support  to  these  data, 
as  does  the  later  work  of  the  same  author,  in  which  the  protein  of 
corn  was  calculated  to  be  only  61  per  cent  utilizable. 

The  above  data  on  the  digestibility  of  maize  are  quite  contrary 
to  those  obtained  for  "roborat,"1  an  albumose-like  preparation. 
"Roborat"  has  been  the  subject  of  considerable  investigation, 
notably  by  Laves,  Loewy  and  Pickart,  Wintgen,  Hoppe,  and  Som- 
merfeld,  the  consensus  of  opinion  being  that  this  commercial  mate- 
rial is  quite  as  well  utilized  as  meat. 

Pertinent  objections  may  be  raised  to  all  the  foregoing  data  on 
the  digestibility  of  the  proteins  of  corn.  The  maize  employed 
contained  considerable  starch,  and  it  is  an  open  question  as  to 
what  extent  the  cells  were  ruptured.  The  unfavorable  influence 
of  these  conditions  has  been  discussed  at  length  in  a  previous  paper.2 
While  these  objections  do  not  apply  to  the  thoroughly  digested 
"roborat,"  still  here  the  protein  has  been  considerably  changed, 
being  present  in  part  as  proteoses. 

Investigations  on  the  pure  unchanged  protein  would  of  course 
be  free  from  these  criticisms.  Maize  contains  several  proteins,3 
one  of  which  is  zein,  an  alcohol-soluble  protein,  which  makes  up 
about  half  the  total  protein.  The  experiments  on  zein  are  prac- 
tically limited  to  those  of  Rockwood  on  dogs.  In  two  experi- 
ments this  observer  found  the  utilization  of  zein  to  be  78  and  90 
per  cent  respectively.  It  should  be  noted  that  the  zein  employed 
by  Rockwood  was  hard  and  not  very  finely  divided,  and  it  is  pos- 
sible that  this  may  have  been  a  factor  contributing  to  the  poor 
utilization. 

Henriques  has  studied  zein  with  reference  to  its  ability  to  main- 
tain nitrogenous  equilibrium  in  rats.  Incidentally  one  notes 
from  his  tables  that  the  zein  varied  in  its  apparent  utilization  be- 
tween 50  and  90  per  cent.  However,  zein,  which  had  been  pre- 
viously subjected  to  tryptic  digestion  was  only  74  per  cent  utilized; 

1  Vor  an  account  of  the  properties  of  this  material  compare  Loewy  and 
Pickart  (see  bibliography). 

2  Cf.  Mendel  and  Fine:  This  Journal,  x,  p.  303.  1911. 

»  See  T.  B.  Osborne:  "Die  Pflanzenproteine,"  Ergebnisse  der  Physiologie, 
x,  p.  47,  1910. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  347 


and  zein  obtained  by  precipitation  from  its  solution  in  alkali 
with  acid  was  but  64  per  cent  digested.  Szumowski's1  observa- 
tions may  be  of  interest  in  this  connection.  A  4  kg.  dog  was  fed 
with  a  mixture  of  100  grams  of  finely  ground  zein,  sugar,  lard  and 
water.  Five  hours  later  the  dog  was  bled  to  death,  and  there 
were  recovered  61  grams  of  zein  from  the  stomach,  18  grams  from 
the  small  intestine  and  6  grams  from  the  large  intestine.  In  an- 
other instance,  a  solution  of  alkali-zein  was  fed,  which  resulted 
in  irritation  of  the  stomach  and  diarrhoea.  It  may  be  that  the 
poor  utilization  obtained  by  Henriques  for  zein  subjected  to  tryptic 
digestion,  and  for  the  zein  previously  dissolved  in  alkali,  is  due  to 
the  production  of  diarrhoea.  Indeed  it  is  well  known  that 
proteose  preparations  from  meat  and  other  sources  may  show 
apparently  poor  utilization  for  this  very  reason. 

The  literature  thus  assembled  would  seem  to  favor  the  view  that 
the  unchanged  protein  of  corn  is  poorly  utilized;  but  it  should  be 
borne  in  mind  that  the  conditions  attending  these  experiments 
have  in  practically  no  instance  been  free  from  objection. 

EXPERIMENTAL  PART. 

Product  Employed. 

The  present  studies  were  confined  to  corn  gluten,2  which  con- 
tained practically  no  starch.  A  large  amount  of  this  substance 
was  obtained  by  Dr.  T.  B.  Osborne,  who  very  kindly  supplied 
all  of  this  material  necessary  for  our  experiments. 

Metabolism  Experiments. 

The  gluten,  ground  to  an  impalpable  powder,  was  fed  to  three 
bitches,  the  usual  method  of  procedure  prevailing.3  Dogs  6  and  7 
(Tables  2  and  3)  in  the  experimental  periods  received  no  other 
nitrogenous  substance  except  the  corn  gluten;  while  dog  5  (Table  1) 
received  two-thirds  of  the  total  nitrogen  as  corn  gluten,  and  the 
remainder  in  the  form  of  meat. 

1  Szumowski:  Zeiischrift  fur  physiologische  Chemie,  xxxvi,  p.  198,  1902. 

2  This  material  contained  7.6  per  cent  nitrogen. 

3  See  Mendel  and  Fine:  This  Journal,  x,  p.  303,  1911. 


348 


Utilization  of  Corn  Proteins 


TABLE  1. 

Corn  Gluten  with  Agar  and  Bone  Ash. 


SUBJECT,  DOG  5 

Weight  at  beginning,  5.7  Kg. 
Weight  at  end,  5.4  Kg. 


PERIOD  I 

(5  days) 
Meat  Feeding 


PERIOD  II 

(4  days) 
Corn  Gluten  and 
Meat  Feeding 


PERIOD  III 

(4  days) 
Meat  Feeding 


Composition  of  daily 
diet  


Meat 

Sugar 

Lard 

Agar 

Bone  ash 

Water 


grams 

160 
20 
20 
3 
7 

100 


grams 


Estimated 
calories  530 


Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per 
cent  


Corn 

gluten 
Meat 
Sugar 
Lard 
Agar 
Bone  ash 
Water 
Estimated 

calories  470 
Corn  gluten 
furnished  66.6 
per  cent  of  the 
total  nitrogen 


43 
50 
20 
25 
3 
7 
175 


Meat 
Sugar 
Lard 
Agar 
Bone  ash 
W  ater 


grams 

150 
20 
20 
3 
7 

100 


Estimated 
calories  520 


Daily  Averages 


Daily  Averages 


Daily  Averages 


4.44 
4.73 
4.89 
+0.16 

13.2 
0.29 
2.22 

94.0 


4.21 
4.72 
4.91 
+0.19 

23.5 
0.51 
2.16 

89.7 


3.80 
4.15 
4.90 
+0.75 

14.5 
0.36 
2.48 

92.7 


TABLE  2. 

Corn  Gluten  with  Agar  and  Bone  Ash. 


SUBJECT,  DOG  6 

Weight  at  beginning,  5.3  Kg. 
Weight  at  end,  5.1  Kg. 

PERIOD  I 

(4  days) 
Meat  Feeding 

PERIOD  II 

(5  days) 
Corn  Gluten 
Feeding 

PERIOD  III 

(5  days) 
Corn  Gluten 
Feeding 

PERIOD  IV 

(5  days) 
Meat  Feeding 

Composition  of  daily 
diet   1 

Nitrogen  output. 
Urine  nitrogen,  gm. . . . 

Total  nitrogen,  gm  

Nitrogen  in  food,  gm . .  . 
Nitrogen  balance,  gm.  . 
Feces. 

Weight,  air  dry,  gm. .  . 

Nitrogen,  gm  

Nitrogen,  per  cent.... 
Nitrogen  utilization, 
per  cent  

grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  3 
Bone  ash  7 
Water  100 

Estimated 
calories  520 

grams 

Corn 

gluten  65 
Sugar  20 
Lard  25 
Agar  3 
Bone  ash  7 
Water  175 
Estimated 
calories  420 

grams 

Corn 

gluten  65 
Sugar  20 
Lard  25 
Agar  3 
Bone  ash  7 
Water  200 
Estimated 
calories  420 

grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  3 
Bone  ash  7 
Water  100 

Estimated 
calories  520 

Daily  Averages  Daily  Averages 

Daily  Averages 

Daily  Averages 

3.89 
4.16 
4.93 
+0.77 

12.2 
0.28 
2.28 

94.3 

4.79 
5.29 
4.95 
-0.34 

26.0 
0.50 
1.92 

89.9 

4.71 
5.17 
4.95 
-0.22 

21.0 
0.46 
2.17 

90.7* 

3.70 
4.06 
4.93 
+0.87 

13.6 
0.36 
2.64 

92.7 

"By  an  accident,  about  half  the  feces  of  this  period  were  charred,  giving  rise  to  possible 
in  analj^sis. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  349 


TABLE  3. 

Corn  Gluten  with  Agar  and  Bone  Ash. 


SUBJECT,  DOQ  7 

Weight  at  beginning,  5.6  Kg. 
Weight  at  end,  4.9  Kg. 


Composition  of  daily 
diet  


Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm. . . . 
Nitrogen  balance,  gm  . . 
Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen,  utilization,  per 
cent  


PERIOD  I 

(4  days) 
Meat  Feeding 


Daily  Averages 


2.98 
3.26 
3.29 
+0.03 

12.5 
0.28 
2.26 

91.4 


PERIOD  II 

(4  days) 
Corn  Gluten 
Feeding 


grams 

grams 

grams 

Meat  100 

Corn 

Meat 

100 

Sugar  20 

gluten 

43 

Sugar 

20 

Lard  20 

Sugar 

20 

Lard 

25 

Agar  3 

Lard 

25 

Agar 

3 

Bone  ash  7 

Agar 

3 

Bone  ash 

7 

Water  100 

Bone  ash 

7 

Water 

100 

Water 

160 

Estimated 

Estimated 

Estimated 

calories  430 

calories 

390 

calories 

470 

Daily  Averages 


3.30 
3.66 
3.27 
-0.39 

19.2 
0.36 
1.86 

89.0* 


PERIOD  III 

(5  days) 
Meat  Feeding 


Daily  Averages 


2.58 
2.81 
3.29 
+0.48 

12.8 
0.23 
1.81 

93.0 


*  By  an  accident,  about  half  the  feces  of  this  period  were  charred,  giving  rise  to  possible  errors 
In  analysis. 

From  the  accompanying  brief  summary  it  is  apparent  that  the 
protein  of  the  corn  gluten  is  only  slightly  less  well  utilized  than  meat 
similarly  fed. 

Summary  of  the  Data  on  Nitrogen  Utilization  (see  Tables  1-3). 


DOG 

CORN  GLUTEN 

MEAT  (AVERAGE) 

per  cent 

per  cent 

5 

89.7 

93.3 

6 

89.9 

93.5 

6 

90.7 

93.5 

7 

89.0 

92.2 

35° 


Utilization  of  Corn  Proteins 


Certain  considerations  lead  us  to  believe  that  under  ideal  con- 
ditions the  utilization  of  corn  would  compare  even  more  favorably 
with  that  of  meat.  The  proportion  of  protein  to  crude  fiber  in 
corn  flour1  is  approximately  8:1.  It  is  probable  therefore  that 
the  corn  gluten  of  our  experiments  had  a  crude  fiber  concentra- 
tion approximating  6  per  cent.  In  the  light  of  experiments  to  be 
reported  in  detail  in  a  subsequent  paper,  it  is  conceivable  that  the 
cellulose,  etc.,  thus  included  in  the  diet  would  in  part  account  for 
the  difference  in  the  coefficients  of  digestibility  of  the  proteins 
of  corn  and  meat. 

TABLE  4. 


Utilization  with  Reference  to  Indigestible  Materials  in  the  Diet.  Daily 

Averages. 


DOG 

PERIOD 

DAYS 

NATURE  OF  INGESTA 

FIBER 
IN  OR 
ADDED 
TO  FOOD 

TOTAL 
INDIGES- 
TIBLE 
MATERIAL 
IN  FOOD 

N 

INTAKE 

N 

UTILIZA- 
TION 

AVER- 
AGE N 
UTILIZA- 
TION 

grams 

grams 

grams 

grams 

per  cent 

per  cent 

5 

XV* 

4 

1  Meat  f 

6 

13 

3.3 

91.6 

6 

xvi* 

4 

>  Bone  ash        5  < 

6 

13 

3.3 

87.7 

J  89.2 

7 

XV* 

4 

J  Agar             2  [ 

6 

13 

3.3 

88.3 

6 

ii 

5 

1  Corn  gluten 

4 

14t 

4.9 

89.9 

6 

iii 

5 

>  Bone  ash        7  I 

4 

14t 

4.9 

90.7 

J  89.9 

7 

ii 

4 

J  Agar              3  [ 

3 

I— « 

CO 

3.3 

89.0 

*  The  details  of  these  experiments  appear  in  a  subsequent  paper  in  this  series, 
t  These  values  are  low  if  anything,  a  certain  amount  of  indigestible  hemicelluloses  probably 
being  present. 


From  Table  4  it  is  apparent  that  meat  diets  containing  amounts 
of  indigestible  materials  comparable  to  those  in  the  corn  gluten 
diets  were  no  more  thoroughly  utilized  than  the  corn  gluten. 
From  this  point  of  view,  the  proteins  of  corn  are  evidently  quite 
thoroughly  utilized. 

There  may  be  some  doubt  as  to  the  validity  of  certain  of  the 
data  in  Tables  2  and  3  as  is  pointed  out  in  foot-notes  to  the  tables; 
but  the  data  are  in  such  close  accord  with  others,  about  which 
there  are  no  such  uncertainties,  that  we  are  inclined  to  accept  them 
as  giving  true  pictures  of  the  actual  state  of  affairs. 

1  See  Atwater  and  Bryant:  U.  S.  Department  of  Agriculture,  Office  of 
Experiment  Stations,  Bull.  28  (revised),  p.  56,  1906. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  351 


A  rather  unsuccessful  attempt  was  made  to  determine  directly 
the  influence  of  indigestible  materials  upon  the  utilization  of  corn 
proteins.  Agar  and  bone  ash  were  omitted  from  the  food  mixtures. 
(See  Table  5.)  A  new  sample  of  gluten  was  employed,  toward 
which  the  animals  acted  very  differently  than  they  did  toward  the 
first  sample.  Dogs  5  and  6  vomited  practically  all  their  food,  and 
dog  7  yielded  results  which  cannot  be  accepted  entirely  without 
question,  since,  as  pointed  out  in  the  foot-note  to  the  table,  a  por- 
tion of  the  food  here  also  was  ejected.  Here  the  utilization  of  the 
corn  protein  is  decidedly  inferior  to  that  of  meat,  and  even  thus,  the 
apparent  utilization  recorded  is  better  than  is  actually  the  case, 
since  not  all  the  food  noted  in  the  table  was  submitted  to  diges- 
tion. Why  the  animals  acted  so  differently  toward  these  two 
samples  of  corn  gluten,  we  are  unable  to  explain.  Although  quite 
finely  ground,  the  second  sample  was  not  an  impalpable  powder, 
and  this  may  account  for  its  low  digestibility  This  explanation 
is  not  especially  satisfactory  to  us 


TABLE  5. 

Corn  Gluten  without  Agar  and  Bone  Ash. 


SUBJECT,  DOG  7 

Weight  at  beginning,  6.6  kg. 
Weight  at  end,  6.6  kg. 


Composition  of  daily  diet 


Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen  per  cent  

Nitrogen  utilization,  per  cent 


PERIOD  XXVII* 

(3  days) 
Corn  Gluten  Feeding 


grams 

grams 

Corn  gluten 

61 

Meat 

150 

Sugar 

25 

Sugar 

25 

Lard 

25 

Lard 

20 

Water 

225 

Water 

100 

Estimated 

Estimated 

calories 

450 

calories 

530 

PERIOD  XXVIII 

(5  days) 
Meat  Feeding 


Daily  Averages 

Daily  Averages 

4.38 

3.60 

4.78 

3.75 

4.76 

4.64 

-0.02 

+0.89 

10.7 

3.8 

0.41 

0.15 

3.82 

3.82 

91.4 

96.9 

|  *  For  the  first  two  days,  the  food  was  greedily  eaten;  on  the  last  day  a  small  part  of  the  food 
had  to  be  forced.   About  one-tenth  of  the  food  of  the  last  day  was  vomited. 


352 


Utilization  of  Corn  Proteins 


From  Table  6  it  is  apparent  that  with  one  exception,  negative 
balance  resulted  in  the  corn  gluten  periods  while  positive  balances 
obtained  throughout  the  meat  periods.  The  one  exception  is  that 
of  Dog  5,  and  in  this  case  it  will  be  recalled  that  one-third  of  the 
daily  nitrogen  was  supplied  by  meat.  Henriques  failed  to  estab- 
lish nitrogenous  equilibrium  with  zein  in  rats 

TABLE  6. 


Average  Daily  Balances.  —  Nitrogen,  in  grams. 


SUBJECT 

TABLE 

GLUTEN  PERIODS 

CORRESPONDING  MEAT 
PERIODS 

Dog  5  

1 

+0.19 

+0.16,  +0.75 

Dog  6  

2 

-0.34,  -0.22 

+0.77,  +0.87 

Dog  7  

3 

-0.39 

+0.03,  +0.48 

Dog  7  

5 

-0.02 

+0.89 

SUMMARY 

Corn  proteins,  partially  purified,  were  somewhat  less  thoroughly 
utilized  than  meat.  Evidence  was  presented  to  indicate  that  this 
small  difference  may  in  great  part  be  attributed  to  the  cell  resi- 
dues remaining  in  the  corn  preparation  employed 

BIBLIOGRAPHY. 

Albertoni  and  Novi:  Archiv  fur  die  gesammte  Physiologie,  lvi,  p.  213> 
1894. 

Erismann:  Zeitschrift  fur  Biologie,  xlii,  p.  672,  1901. 

Grandeau,  LeClerc,  and  Ballacey:  Quoted  from  Szumowski:  Zeit- 
schrift fur  physiologische  Chemie,  xxxvi,  p.  198,  1902. 

Henriques:  Zeitschrift  filr  physiologische  Chemie,  lx,  p.  105,  1909. 

Hoppe:  Milnchener  medizinische  Wochenschrift,  xlix,  p.  479,  1902. 

Laves:  Milnchener  medizinische  Wochenschrift,  p.  1339,  1900. 

Loewy  und  Pickart:  Deutsche  medizinische  Wochenschrift,  p.  821,  1900. 

Malfatti:  Sitzungs-Berichte  der  Wiener  Academie,  xc,  (3),  p.  323,  1884. 

Merrill:  Maine  Agricultural  Experiment  Station,  Bull.  131,  1906;  Ibid., 
Bull.  158,  1908. 

Rockwood:  American  Journal  of  Physiology,  xi,  p.  355,  1904. 

Rubner:  Zeitschrift  fur  Biologie,  xv,  p.  115,  1879. 

Sommerfeld:  Archiv  fiir  Kinder  heilkunde,  xxxvi,  p.  341,  1903. 

WintgExNt:  Zeitschrift  fiir  Untersuchung  der  Nahrungs-und  Genussmittel, 
v,  p.  289,  1902. 


Reprinted  from  The  Journal  or  Biological  Chemistry,  Vol.  X,  No.  6,  1912 


STUDIES  IN  NUTRITION. 
IV.    THE  UTILIZATION  OF  THE  PROTEINS  OF  THE  LEGUMES. 

By  LAFAYETTE  B.  MENDEL  and  MORRIS  S.  FINE. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  September  25,  1911.) 

CONTENTS. 


Earlier  studies   433 

Experimental  part   435 

Products  employed   435 

Metabolism  experiments   437 

Soy  bean   437 

White  bean   446 

Crude  bean  protein  ,   448 

Phaseolin   454 

Pea  globulin   454 

Nitrogen  balances   456 

Summary   457 


EARLIER  STUDIES. 

The  literature  on  this  subject  has  been  so  adequately  reviewed 
by  Wait,  that  only  the  most  cursory  consideration  of  the  earlier 
work  need  find  place  here.  In  experiments  on  a  man,  Hoffmann 
found  the  nitrogen  of  a  diet  of  lentils,  bread  and  potatoes  to  be 
58  per  cent  available,  against  a  utilization  of  82  per  cent  for  the 
nitrogen  of  meat.  Woroschiloff  compared  the  utilization  of  the 
protein  of  peas  with  that  of  meat  protein.  In  three  cases  the  meat 
protein  was  90,  92,  and  96  per  cent  utilized  against  83,  88,  and  90 
per  cent  for  the  digestibility  of  the  protein  of  the  peas.  Striimpell 
found  the  nitrogenous  constituents  of  "leguminose" — a  finely 
ground  commercial  preparation,  consisting  of  a  mixture  of  lentils, 
peas  and  rye — to  be  90  per  cent  available,  against  a  utilization  of 


433 


434 


Utilization  of  Legume  Proteins 


but  60  per  cent  in  an  experiment  with  unground  lentils.  Rubner 
has  pointed  out  that  in  the  experiments  of  both  Woroschiloff  and 
Strumpell,  materials  other  than  legumes  were  eaten,  and  these 
accessories  may  have  exerted  a  favorable  influence.  Accordingly, 
Rubner  conducted  two  experiments  with  thoroughly  cooked  hulled 
peas  which  were  the  only  food  consumed.  The  utilization  was  72 
to  83  per  cent.  In  Malfatti's  experiments,  peas  were  86  per  cent 
utilized  and  Potthast  found  lentils  to  be  74  per  cent  digested. 
In  an  experiment  by  Prausnitz,  white  beans,  soaked  for  several 
hours  and  then  cooked  till  soft,  yielded  70  per  cent  available 
nitrogen.  Erismann  found  the  nitrogen  of  peas  to  be  80  per  cent 
digested.  Richter  obtained  a  utilization  of  90  per  cent  for  the 
nitrogen  of  peas  cooked  in  distilled  water,  against  83  per  cent  when 
hard  water  was  employed  in  the  process  of  cooking.  Under  the 
latter  condition  particles  of  apparently  unchanged  peas  were  ob- 
served in  the  feces.  The  poor  digestibility  of  the  peas  cooked  in 
hard  water  is  attributed  in  part  to  the  formation  of  difficultly 
digestible  alkali  earth  albuminates,  and  in  part  to  the  digestive 
disturbance  due  to  the  magnesium  salts  in  the  water.  Snyder 
reported  a  utilization  of  80  per  cent  for  the  protein  of  peas,  and 
obtained  a  similar  result  with  beans.  In  their  experiments  with 
the  Maine  lumbermen,  Woods  and  Mansfield  estimated  the  pro- 
tein of  beans  to  be  at  least  78  per  cent  utilizable,  and  an  average 
digestibility  of  65  per  cent  is  reported  in  Oshima's  compilation  of 
Japanese  investigations.  In  a  very  thorough  study,  Wintgen 
found  the  average  coefficients  of  digestibility  of  lentils,  beans,  and 
peas  to  be  78,  80  and  86  per  cent  respectively.  Wintgen's  results 
are  in  accord  with  those  obtained  in  an  extensive  investigation  by 
Wait,  in  which  a  utilization  of  77  to  78  per  cent  was  obtained  for 
bean  protein,  and  70  to  83  per  cent  for  cow  pea  protein. 

In  commenting  upon  this  literature  one  can  but  reiterate  the 
statements  made  in  a  previous  paper1  of  this  series,  and  point  out 
the  necessity  for  studying  the  utilization  of  the  isolated  protein,  or 
material  in  which  the  protein  is  more  readily  accessible  to  the 
digestive  juices. 


1  Mendel  and  Fine:  This  Journal,  x,  p.  303;  1911. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  435 


EXPERIMENTAL  PART. 


Products  Employed. 


1 .  Soy  Bean.1  This  material  was  an  impalpable  yellow  pow- 
der, which  betrayed  no  cellular  structure  under  the  microscope. 
In  respect  to  consistency,  it  would  thus  appear  to  be  ideal  for 
digestion  experiments.  As  may  be  observed  from  the  accompany- 
ing analysis,2  the  soy  bean  offers  several  points  of  interest : 


Its  content  of  protein  and  fat  far  exceeds  that  of  any  other 
legume,  which  condition  seems  to  have  been  appreciated  in  Japan ; 
for,  according  to  Oshima,  it  is  next  to  rice  in  importance  in  the 
Japanese  dietary.3  In  addition  to  cane  sugar,  the  presence  of 
galactans  and  of  pentosans  has  been  detected  by  Schulze  and  his 
collaborators.4  The  soy  bean  does  not  give  the  ordinary  iodine 
test  for  starch.5 

2.  White  Bean.  This  was  the  ordinary  white  bean  of  com- 
merce. 

3.  Crude  Bean  Protein.  Experiments  with  the  ordinary 
white  bean  are  subject  to  the  same  criticism  as  has  been  offered  in 
connection  with  the  work  of  previous  investigators.  The  attempt 
was  here  made  to  thoroughly  rupture  the  cells  and  dissolve  and 
wash  away  the  starch.    The  method  in  brief  was  as  follows:  about 

1  Mr.  M.  F.  Deming  of  the  Cereo  Company,  Tappan,  N.  Y.,  very  kindly 
contributed  this  material. 

2  Reported  by  Ruhrah:  Journal  of  the  American  Medical  Association,  liv, 
p.  1664,  1910. 

3  Oshima,  (see  bibliography)  gives  an  interesting  account  of  the  various 
soy  bean  preparations,  which  are  common  articles  of  diet  in  Japan. 

4  For  the  literature,  see  Schulze  and  Godet:  Zeitschrift  fur  physiologischc 
Chemie,  lxi,  p.  279,  1909. 

5Cf.  Oshima:  loc.  cit.  p.  26. 


per  cent 


Protein 

Fat. 


44.6 
19.4 
9.3 
4.2 
2.3 
5.3 
14.8 


Cane  sugar  

Mineral  matter  

Crude  fiber  

Moisture  

Non-nitrogenous  extract 


436  Utilization  of  Legume  Proteins 

5  pounds  of  finely  ground  hulled  beans1  were  mixed  with  water 
and  heated  in  a  glycerol  bath.  After  the  mixture  had  been  held 
near  100°  C.  for  about  an  hour,  the  thin  mush  which  had  formed 
was  cooled  below  75°  C.  and  a  glycerol  extract  of  malt  diastase 
added,  as  a  result  of  which,  after  a  few  minutes,  starch  could  no 
longer  be  detected  with  iodine  in  a  test-tube  trial.  The  material 
thus  obtained  was  washed  by  decantation  and  the  water  driven  off 
by  heat  until  about  20  per  cent  was  made  up  of  solid  matter.  The 
resulting  preparation  was  a  thick  mush,  which  could  be  conven- 
iently pressed  into  cakes  and  preserved  frozen.  Although  no 
iodine  test  for  starch  was  obtained  in  a  test-tube  trial,  nevertheless, 
when  a  sample  treated  with  this  reagent  was  examined  under  the 
microscope,  not  infrequently  starch  grains  were  observed  within 
cells,  which  had  apparently  not  in  any  way  been  affected  by  the 
treatment  to  which  they  had  been  subjected.  This  insufficient 
rupture  undoubtedly  accounts  for  the  incomplete  conversion  of 
the  starch. 

'   Analysis  of  Crude  Bean  Protein  (calculated  for  anhydrous  material). 

per  cent 


Protein  (N  X  6.25)   51 . 1 

Sugar  from  insoluble  carbohydrate  (by  hydrolysis)  28.9 

Sugar  from  soluble  carbohydrate  (by  hydrolysis)   2.4 

Ash   2.6 

Ether  extract*   4.0 

Crude  fiber  (by  difference)   11 .0 


♦Estimated  from  Atwater  and  Bryant:  U.  S.  Department  of  Agriculture,  Bull.  28  (Revised), 
p.  65,  1906.   Material  was  not  available  for  analysis. 

Attention  is  called  to  the  fact  that  a  barley  protein  preparation2 
with  approximately  the  same  concentration  of  protein  contained 
practically  no  cellular  structure  or  starch,  yet  yielded  20  per  cent 
of  carbohydrate  by  hydrolysis.  The  latter  was  believed  to  be 
hemicelluloses.  It  is  thus  probable  that  a  not  inconsiderable  portion 
of  the  "  carbohydrate  by  hydrolysis"  of  the  above  analysis  was  in 
reality  also  made  up  of  hemicelluloses.3 

1  Furnished  by  Mr.  Deming  who  also  prepared  a  considerable  portion  of 
the  crude  bean  protein  for  us  according  to  the  method  outlined. 

2  Mendel  and  Fine:  This  Journal,  x,  p.  340,  1911. 

3  At  the  time  of  proof  reading  we  learn  through  a  private  communication 
from  Prof.  E.  Schulze  that  hulled  beans,  phaseolus  vulgaris,  contain  12.9 
per  cent  hemicellulose.  We  estimate  the  hemicellulose  concentration  of 
our  preparation  at  approximately  25  per  cent. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  437 

4.  Phaseolin.  This  material  was  very  kindly  furnished  by 
Dr.  T.  B.  Osborne.  It  was  dried,  ground  to  an  impalpable  powder, 
and  found  by  analysis  to  contain  13  per  cent  of  nitrogen. 

5.  Pea  Globulin.  This  material  was  prepared  as  follows: 
dried  peas  were  finely  ground  and  repeatedly  extracted  with  10 
per  cent  NaCl  solution.  The  perfectly  clear  extract  thus  obtained 
was  saturated  with  ammonium  sulphate,  the  resulting  precipi- 
tate being  collected  on  a  filter  paper,  suspended  in  a  small  amount 
of  water  to  which  toluene  had  been  added  and  dialyzed  for  about 
two  weeks,  that  is,  until  free  from  sulphates.  Part  of  the  prepar- 
ation was  obtained  by  dialyzing  the  saline  extract,  thus  avoiding 
the  necessity  of  the  precipitation  with  ammonium  sulphate.  The 
resulting  precipitate  was  dried  at  40°  to  50°  C,  ground  to  an  im- 
palpable powder  and  found  by  analysis  to  contain  16  per  cent  of 
nitrogen. 

Metabolism  Experiments. 

Soy  Bean.  Man,1  Table  1:  The  ordinary  routine  was  followed : 
a  fore  period  (preceded  by  a  three  day  adjustment  period),  during 
which  a  mixed  diet  was  consumed;  experimental  period,  in  which 
over  90  per  cent  of  the  nitrogen  ingested  was  furnished  by  soy 
bean;  and  an  after  period  essentially  like  the  fore  period.  The 
character  of  the  diet  is  outlined  below: 


Character  of  Diet. 


PRELIMINARY  AND 
FORE  PERIODS 

EXPERIMENTAL 
PERIOD 

AFTER  PERIOD 

Daily  Averages 

Daily  Averages 

Daily  Averages 

grams 

grams 

grams 

Cracker  

70 

70 

Egg  

100 

200 

Peanut  butter  

75 

Meat  

140 

190 

Soy  bean  

165 

Potato  

100 

120 

Tomato  

250 

375 

200 

Apple  

200 

200 

200 

Orange  

180 

180 

180 

Milk  

60 

60 

60 

Sugar  

130 

140 

130 

Butter  

50 

100 

90 

Cereal  coffee,  tea  

600 

600 

1  The  subject  was  one  of  us  (M.  S.  F.)  twenty-four  years  of  age,  leading 
the  usual  active  life  of  the  laboratory. 


438  Utilization  of  Legume  Proteins 


As  will  be  observed,  during  the  experimental  period  the  cracker, 
egg,  meat,  and  nut  butter  were  completely  replaced  by  soy  bean, 
which  furnished  91  per  cent  of  the  total  nitrogen  intake  of  this 
period.  The  daily  nitrogen  and  calorific  intakes  in  these  periods 
were  fairly  constant,  averaging  about  12.6  grams  and  2500  calories, 
respectively.  The  soy  bean  was  boiled  in  water  for  one-half 
hour,  salted  to  taste,  and  the  tomatoes  thoroughly  incorporated 
into  the  resulting  mush.  The  palatability  of  the  mixture  was  still 
further  increased  by  the  addition  of  a  very  small  amount  of  pap- 
rika. On  the  whole  it  may  be  said  that  this  fare  proved  quice 
agreeable,  no  unpleasant  symptoms  appearing  throughout  the 
period  of  six  days. 

TABLE  1. 


Soy  Bean. 


SUBJECT,  MAN 

PERIOD  I 

PERIOD  II 

PERIOD  III 

'  Weight  at  beginning,  56.8  Kg. 

(5  clays) 

(6  days) 

(5  days) 

Weight  at  end,  56.6  Kg. 

Mixed  Diet 

Soy  Bean 

Mixed  Diet 

Meat,  egg, 

Soy  bean, 

Meat,  egg,  po- 

nut butter, 

fruit,  etc., 

tato,  fruit, 

potato, 

90.5  per  cent 

etc.  (No  nut 

Composition    of  daily 

fruit,  etc. 

total  nitro- 

butter). 

gen  supplied 

by  soy  bean. 

Estimated 

Estmated 

Estimated 

calories  2400 

calories  2400 

calories  2600 

Nitrogen  output. 

Daily  Averages 

Daily  Averages 

Daily  Averages 

Urine  nitrogen,  gm  

9.56 

10.81 

9.69 

Total  nitrogen,  gm  

11.11 

12.72 

11.16 

Nitrogen  in  food,  gm   

12.78 

12.93 

12.22 

Nitrogen  balance,  gm. . . . 

+1.67 

+0.21 

+1.06 

Feces. 

Weight  air  dry,  gm  

25.0 

26.6 

25.2 

Nitrogen,  gm  

1.55 

1.91 

1.47 

Nitrogen,  per  cent  

6.18 

7.15 

5.88 

Nitrogen  utilization,  per 

cent  

87.9 

85.3 

88.0 

The  subject  felt  in  excellent  condition  throughout  the  entire 
experiment.  Defecation  took  place  regularly  every  morning  and 
no  diarrhoea  occurred. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  439 

It  will  be  observed  from  Table  1,  that  the  soy  bean  nitrogen  is 
distinctly  (if  only  slightly)  less  well  utilized  than  that  of  the  preced- 
ing and  succeeding  mixed  diets.  The  nitrogen  concentration  of 
the  feces  of  the  soy  bean  period  is  higher  than  in  any  other  experi- 
ment on  this  subject  which  indicates  that  some  soy  bean  protein 
escaped  absorption. 

Soy  Bean.  Dogs  (with  agar  and  bone  ash) — Tables  2  to  4: 
Dog  1,  Table  2,  was  fed  with  a  mixture  of  soy  bean,  lard,  agar, 
bone  ash  and  water.  It  was  heated  on  the  water  bath  for  four  to 
six  hours,  the  purpose  being  to  thoroughly  "  hydrate"  the  material, 
which,  as  fed  to  the  animal,  was  a  thick  mush. 

Dogs  5  and  7,  Tables  3  and  4,  were  fed  with  similar  ingredients 
and  sugar  in  addition.  The  mixture,  including  the  water,  was  not 
heated,  but  allowed  to  stand  over  night,  after  which  the  material 
appeared  to  be  thoroughly  "hydrated." 

The  plan  of  experimentation  differed  in  no  particular  from  those 
previously  followed,  and  we  may  therefore  proceed  directly  to  an 
examination  of  the  tables,  which  contain  all  the  essential  details. 

In  the  dog,  the  soy  bean  was  in  every  case  strikingly  less  well  util- 
ized than  the  meat  fed  under  similar  experimental  conditions,  and 
one  also  notes  the  persistently  higher  nitrogen  concentration  of 
the  feces  of  the  soy  bean  periods  as  compared  with  that  of  the  meat- 
feces.  This  is  an  indication,  as  noted  above,  that  some  soy  bean 
protein  has  probably  escaped  digestion. 


44°  Utilization  of  Legume  Proteins 

TABLE  2. 


Soy  Bean  with  Agar  and  Bone  Ash. 


SUBJECT,  DOG  1 

Weight  at  beginning,  15.0  Kg. 
Weight  at  end,  14.6  Kg. 

PERIOD  III 

(5  days) 
Meat  Feeding 

PERIOD  ivf 

(5  days) 
Soy  Bean  Feeding 

PERIOD  V 

(4  days) 
Meat  Feeding 

Composition    of  daily 
diet  ' 

iv  til  uyvii  viAiiputi, 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm. . . . 
Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per 
cent  

grams 

Meat  300 
Lard  60 
Agar*  5 
Bone  ash  15 
Water  300 
Estimated 
calories  1070 

grams 

Soy  bean  147 
Lard  60 
Agar  5 
Bone  ash  15 
Water  500 
Estimated 
calories  1110 

grams 

Meat  300 
Lard  60 
Agar  5 
Bone  ash  15 
Water  300 
Estimated 
calories  1070 

Daily  Averages 

Daily  Averages 

Daily  Averages 

10.07 
10.60 
10.38 
-0.22 

29.4 
0.53 
1.79 

95.0 

9.25 
10.94 
10.44 
-0.50 

55.6 
1.69 
3.05 

83.8 

8.81 
9.38 
10.44 
+1.06 

29.5 
0.57 
1.95 

94.5 

*  On  the  first  two  days  of  the  period,  the  "indigestible"  was  represented  by  20  grams  bone 
ash.  This  produced  brittle  feces,  hence  in  the  remaining  three  days  agar  and  bone  ash  were  em- 
ployed as  noted  in  the  table. 

t  Forced  feeding  necessary  throughout  the  period— no  vomiting. 

TABLE  3.  . 

Soy  Bean  with  Agar  and  Bone  Ash. 

SUBJECT,   DOG  5 

Weight  at  beginning,  5.2  Kg. 
Weight  at  end,  5.2  Kg. 

PERIOD  VI 

(4  days) 
Meat  Feeding 

PERIOD  VII* 

(4  days) 
Soy  Bean  Feeding 

PERIOD  VIII 

(5  days) 
Meat  Feeding 

Composition    of    daily  ( 
diet  | 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per 

grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  3 
Bone  ash  7 
Water  100 
Estimated 
calories  510 

grams 

Soy   bean  69 
Sugar  20 
Lard  25 
Agar  3 
Bone  ash  7 
Water  200 
Estimated 
calories  570 

grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  3 
Bone  ash  7 
Water  100 
Estimated 
calories  510 

Daily  Averages 

Daily  Averages 

Daily  Averages 

3.71 
4.11 
4.93 
+0.82 

15.5 
0.40 
2.60 

91.8 

3.01 
4.26 
4.90 
+0.64 

34.0 
1.25 
3.67 

74.5 

4.09 
4.44 
4.80 
+0.36 

15.0 
0.35 
2.32 

92.7 

Cystitis  developed  but  was  cured  in  the  course  of  two  days  by  means  of  AgNOj  solution. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  441 


TABLE  4. 

Soy  Bean  with  Agar  and  Bone  Ash. 


SUBJECT,  DOG  7 

PERIOD  V 

PERIOD  VI 

PERIOD  VII 

Weight  at  beginning,  4.9  Kg. 

(3  days) 

(6  days) 

(5  days) 

Weight  at  end,  4.6  Kg. 

Meat  Feeding 

Soy  Bean  Feeding 

Meat  Feeding 

QTdTilS 

QTC177XS 

QTQ7718 

Meat  100 

Soy  bean  47 

Meat  100 

Sugar  20 

Sugar  20 

Sugar  20 

Lard  20 

Lard  25 

Lard  20 

Composition    of  daily 

Agar  3 

Agar  3 

Agar  3 

diet  1 

Bone  ash  7 

Bone  ash  7 

Bone  ash  7 

Water  100 

Water  175 

Water  100 

Estimated 

Estimated 

Estimated 

calories  430 

calories  490 

calories  430 

Nitrogen  output. 

Daily  Averages 

Daily  Averages 

Daily  Averages 

Urine  nitrogen,  gm  

O  AO 

Z.bo 

o  oo 

2.00 

9  «Q 

z .  oy 

6  .  IHt 

9  7Q 

z .  /y 

Nitrogen  in  food,  gm  

3.29 

3.34 

3.20 

Nitrogen  balance,  gm. .  . . 

+0.40 

+0.30 

+0.41 

Feces. 

Weight  air  dry,  gm  

12.7 

20.5 

12.8 

Nitrogen,  gm  

0.26 

0.66 

0.24 

Nitrogen,  per  cent  

2.04 

3.22 

1.86 

Nitrogen  utilization,  per 

cent  

92.1 

80.2 

92.6 

Soy  Bean.  Dogs  (without  agar  and  bone  ash) — Tables  5  to  18: 
These  experiments  were  conducted  in  essentially  the  same  manner 
as  those  just  reported,  except  that  the  indigestible  adjuvants — 
agar  and  bone  ash — were  omitted.  Tables  8  to  10  contain  the 
results  of  trials  instituted  after  the  intestinal  tract  had  been  sub- 
jected to  a  thorough  treatment  with  indigestible  non-nitrogenous 
materials,  the  purpose  being  to  remove  as  far  as  possible  the  accu- 
mulated intestinal  debris. 

Proceeding  directly  to  a  study  of  the  tables,  we  again  note  the 
poor  utilization  of  the  soy  bean  nitrogen.  A  fuller  discussion  of 
these  data  with  a  consideration  of  the  attending  conditions  will 
be  offered  below1  in  connection  with  the  discussion  of  the  results 
obtained  with  the  crude  bean  protein. 

In  Oshima's  compilation  one  notes  that  certain  soy  bean  products 
{e.g.,  tofu)  are  as  much  as  96  per  cent  utilizable.  Tofu,  however, 
is  probably  of  an  albumose  nature  and  such  favorable  results  should 
be  correspondingly  interpreted. 

1  P.  452. 


442  Utilization  of  Legume  Proteins 

TABLE  5. 


Soy  Bean  without  Agar  or  Bone  Ash. 


SUBJECT,    DOG  5 

Weight  at  beginning,  5.9  Kg. 
Weight  at  end,  6.0  Kg. 

PERIOD  XX 

(4  days) 
Meat  Feeding 

r  Hi IXIKJU  AAl 

(5  days) 
Soy  Bean  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Meat  150 
Sugar  25 
Lard  20 
Water      .  100 
Estimated 

calories  530 

grams 

Soy  bean  64 
Sugar  25 
Lard  20 
Water  225 
Estimated 

calories  530 

Daily  Averages 

Daily  Averages 

4.08 
4.30 
4.59 
+0.29 

4.5 
0.22 
4.95 
95.2 

3.96 
4.66 
4.61 
-0.05 

17.8 
0.70 
3.90 

85.0 

One-quarter  to  one-half  of  the  food  forced  each  day. 

TABLE  6. 

Soy  Bean  without  Agar  or  Bone  Ash. 

SUBJECT,  DOG  6 

Weight  at  beginning,  6.1  Kg. 
Weight  at  end,  6.3  Kg. 

PERIOD  XXI 

(4  days) 
Meat  Feeding 

PERIOD  XXII 

(5  days) 
Soy  Bean  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 
Total  nitrogen,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Meat  150 
Sugar  25 
Lard  20 
Water  100 
Estimated 

calories  530 

grams 

Soy  bean  64 
Sugar  25 
Lard  20 
Water  225 
Estimated 

calories  530 

Daily  Averages 

Daily  Averages 

3.55 
3.77 
4.59 
+0.82 

3.5 
0.22 
6.34 
95.2 

3.42 
4.16 
4.61 
+0.45 

18.2 
0.74 
4.04 

84.0 

Lafayette  B.  Mendel  and  Morris  S.  Fine  443 

TABLE  7. 


Soy  Bean  without  Agar  or  Bone  Ash. 


SUBJECT,  DOG  7 

Weight  at  beginning,  5.9  Kg. 
Weight  at  end.  6.3  Kg. 

PERIOD  XX 

(4  days) 
Meat  Feeding 

PERIOD  XXI 

(5  days) 
Soy  Bean  Feeding 

Nitrogen  output. 
Urine  nitrogen,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

grams 

Meat  150 
Sugar  25 
Lard  20 
Water  100 
Estimated 
calories  530 

grams 

Soy  bean  64 
Sugar  25 
Lard  20 
Water  225 
Estimated 

calories  530 

Daily  Averages 

Daily  Averages 

3.55 
3.74 
4.59 
+0.85 

3.2 
0.19 
5.93 
95.8 

3.41 
4.21 
4.61 
+0.40 

16.2 
0.80 
4.91 

82.8 

TABLE  8. 

Soy  Bean  without  Agar  or  Bone  Ash. 

SUBJECT,  DOG  5 

Weight  at  beginning,  6.2  Kg. 
Weight  at  end,  6.2  Kg. 

PERIOD  XXVI* 

(3  days) 
Soy  Bean  Feeding 

PERIOD  XXVIII 

(5  days) 
Meat  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output 

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm.*  

Nitrogen,  gm  

Nitrogen,  per  cent  

grams 

Soy  bean  64 
Sugar  25 
Lard  20 
Water  225 
Estimated 
calories  530 

grams 

Meat  150 
Sugar  25 
Lard  20 
Water  100 
Estimated 

calories  530 

Daily  Averages 

Daily  Averages 

3.66 
4.31 
4.61 
+0.30 

18.5 
0.65 
3.52 

85.9 

3.68 
3.84 
4.64 
+0.80 

3.4 
0.16 
4.62 
96.6 

*  About  half  of  the  food  forced  each  day. 


444  Utilization  of  Legume  Proteins 

TABLE  9. 


Soy  Bean  without  Agar  or  Bone  Ash. 


SUBJECT,  DOG  6 

Weight  at  beginning  6.6  Kg. 
Weight  at  end,  6.6  Kg. 

PERIOD  XXVII 

(3  days) 
Soy  Bean  Feeding 

PTRTflTl  YTTY 
jrSU±\l.\JU  AAiA 

(5  days) 
Meat  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

Soy  bean  64 
Sugar  25 
Lard  20 
Water  225 
Estimated 
calories  530 

Meat  150 
Sugar  25 
Lard  20 
Water  100 
Estimated 

calories  539 

Daily  Averages 

Daily  Averages 

3.45 
4.06 
4.61 
+0.55 

16.7 
0.61 
3.64 

86.8 

3.35 
3.59 
4.64 
+1.05 

4.4 
0.24 
5.42 
94.9 

TABLE  10. 

Soy  Bean  without  Agar  or  Bone  Ash. 

SUBJECT,  DOG  7 

Weight  at  beginning,  6.6  Kg. 
Weight  at  end,  6.6  Kg. 

PERIOD  XXVI 

(4  days) 
Soy  Bean  Feeding 

PERIOD  XXVIII 

(5  days) 
Meat  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 
Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Soy  bean  64 
Sugar  25 
Lard  20 
Water  225 
Estimated 
calories  530 

grams 

Meat  150 
Sugar  25 
Lard  20 
Water  100 
Estimated 

calories  530 

Daily  Averages 

Daily  Averages 

3.78 
4.35 
4.61 
+0.26 

15.9 
0.57 
3.60 

87.6 

3.60 
3.75 
4.64 
+0.89 

3.8 
0.15 
3.82 
96.9 

Lafayette  B.  Mendel  and  Morris  S.  Fine  445 

TABLE  11. 


Soy  Bean  without  Agar  or  Bone  Ash. 


SUBJECT,  DOG  5 

Weight  at  beginning,  6.0  Kg. 
Weight  at  end,  5.9  Kg. 

PERIOD  XVI 

(4  days) 
Meat  Feeding 

PERIOD  XVII 

(4  days) 
Soy  Bean  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Meat  100 
Sugar  25 
Lard  20 
Water  150 
Estimated 
calories  450 

grams 

Soy  bean  46 
Sugar  25 
Lard  20 
Water  225 
Estimated 
calories  460 

Daily  Averages 

Daily  Averages 

2.77 
2.79 
3.28 
+0.49 

0.4 
0.02 
5.32 
99.4* 

2.83 
3.48 
3.31 
-0.17 

14.0 
0.65 
4.67 

80.2 

*  Feces  evidently  separated  imperfectly. 

TABLE  12. 

Soy  Bean  without  Agar  or  Bone  Ash. 

SUBJECT,  DOG  6 

Weight  at  beginning,  6.2  Kg. 
Weight  at  end,  6.1  Kg. 

PERIOD  XVII 

(4  days) 
Meat  Feeding 

PERIOD  XVIII 

(4  days) 
Soy  Bean  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Feces. 

Weight  air  dry.  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Meat  100 
Sugar  25 
Lard  20 
Water  150 
Estimated 
calories  450 

grams 

Soy  bean  46 
Sugar  25 
Lard  20 
Water  225 
Estimated 

calories  460 

Daily  Averages 

Daily  Averages 

2.41 
2.54 
3.28 
+0.74 

1.9 
0.13 
7.03 
96.0 

2.49 
3.21 
3.31 
+0.10 

14.0 
0.72 
5.15 

79.3 

446  Utilization  of  Legume  Proteins 


TABLE  13. 


Soy  Bean  without  Agar  or  Bone  Ash. 

SUBJECT,  DOG  7 

Weight  at  beginning,  6.0  Kg. 
Weight  at  end,  5.9  Kg. 

PERIOD  XVI 

(4  days) 
Meat  Feeding 

PERIOD  XVII 

(4  days) 
Soy  Bean  Feeding 

Composition  of  daily  diet   < 

Nitrogen  output. 

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Meat  100 
Sugar  25 
Lard  20 
Water  150 
Estimated 
calories  450 

grams 

Soy  bean  46 
Sugar  25 
Lard  20 
Water  225 
Estimated 
calories  460 

Daily  Averages 

Daily  Averages 

2.85 
2.95 
3.28 
+0.33 

1.5 
0.1j0 
7.06 
96.8 

2.83 
3.37 
3.31 
-0.06 

11.0 
0.54 
4.77 

83.8 

White  Bean.  Man,  Table  H:  The  original  intention  was  to 
investigate  the  digestibility  of  hulled  beans  which  had  been  finely 
ground  and  thoroughly  cooked,  and  in  which  the  starch  had  in 
great  part  been  dissolved  by  an  amylolytic  preparation.  This 
mixture,  however,  produced  such  violent  nausea  that  a  successful 
experiment  with  it  was  entirely  out  of  the  question.  Indeed  other 
workers,  notably  Strumpell,  have  reported  similar  difficulties  with 
bean  experiments. 

However,  the  subject  was  unwilling  to  have  the  fore  period  stand 
for  naught,  so  the  experiment  was  carried  through  using  ordinary 
unhulled  beans,  which  were  cooked  or  baked  in  the  usual  way. 
In  this  form  the  beans  were  far  from  unpalatable.  The  character 
of  the  dietary  employed  in  this  experiment  is  given  below: 


Lafayette  B.  Mendel  and  Morris  S.  Fine  447 


PRELIMINARY  AND 
PORE  PERIODS 

EXPERIMENTAL 
PERIOD 

Dally  Averages 

Dally  Averages 

grams 

grams 

70 

35 

Egg  

200 

90 

Meat  

200 

Beans  

230 

100 

250 

300 

Apple  

200 

200 

Orange  

180 

180 

120 

140 

Milk  

60 

60 

Sugar  

120 

130 

Butter  

75 

80 

Cereal  coffee,  tea  

600 

600 

TABLE  14. 


White  Bean. 


SUBJECT,  MAN 

Weight  at  beginning,  56.4  Kg. 
Weight  at  end,  56.0 

PERIOD  I 

(4  days) 
Mixed  Diet 

PERIOD  II 

(4  days) 
White  Bean 

Composition  of  daily  diet  < 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight,  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

Meat,  egg,  pota- 
to, fruit,  etc. 

Estimated 
calories  2600 

Beans,  egg,  fruit, 
etc. 

68.2  per  cent  of 
total  nitrogen 
supplied  by  the 
beans. 

Estimated 
calories  2700 

Daily  Averages 

Dally  Averages 

9.19 
10.90 
12.25 
+1.35 

26.2 
1.71 
6.54 

86.0 

8.00 
10.70 
12.20 
+1.50 

44.2 
2.70 
6.11 

77.9 

The  plan  of  the  experiment  differed  in  no  essential  from  those 
on  man  already  reported.  The  preliminary  period  of  adjustment 
was  only  one  day  in  duration,  and  the  after  period  was  entirely 


443 


Utilization  of  Legume  Proteins 


omitted.  The  results  are  in  general  accord  with  those  obtained 
by  previous  observers. 

The  factors  which  probably  contribute  to  the  unfavorable  util- 
ization of  the  protein  of  beans  have  already  been  discussed.1 
The  plan  was  conceived  of  avoiding  these  unfavorable  influences 
as  far  as  possible,  and  to  that  end,  as  already  described,2  hulled 
and  powdered  beans  were  thoroughly  cooked,  and  the  greater  part 
of  the  starch  removed.    Experiments  with  this  material  follow. 

Crude  Bean  Protein3 — Dogs,  Tables  15  to  21:  The  material 
was  not  dried;  but  when  in  the  stage  of  evaporation  the  proportion 
of  solid  matter  became  about  20  per  cent,  it  was  pressed  into  pack- 
ages containing  the  daily  supply  of  protein,  and  preserved  frozen 
until  ready  for  use.  In  some  cases,  no  additional  water  was  given, 
as  the  moisture  of  the  product  sufficed.  Such  details  may  be 
most  readily  learned  from  the  tables. 

In  the  experiments  reported  in  Tables  15  to  17,  the  food  mixture 
was  of  the  consistency  of  putty  and  made  a  rather  large  volume  of 
material.  The  animals  experienced  some  difficulty  in  chewing 
because  of  the  unusual  consistency,  but  in  spite  of  this  Dogs  6 
and  7  appeared  to  relish  the  fare;  and  even  Dog  5,  upon  whom 
forced  feeding  had  to  be  practiced,  did  not  seem  to  find  the  food 
especially  repellent. 

It  was  thought  that  possibly  this  large  mass  of  food  overburdened 
the  digestive  tract  thus  accounting  for  the  rather  poor  utilization, 
and  hence  experiments  reported  in  Tables  18  to  20  were  instituted, 
where  the  food  nitrogen  was  only  two-thirds  as  great  as  in  the  pre- 
ceding three  experiments. 

In  all  the  above  experiments,  agar  and  bone  ash  were  included 
in  the  diet,  and  it  was  therefore  desired  to  eliminate  the  influence 
of  these  "indigestibles"  for  comparison.  Hence  the  experiment 
recorded  in  Table  21. 

1  Cf.  Mendel  and  Fine:  This  Journal,  x,  p.  305,  1911. 

2  Pp.  435-436. 

3  As  far  as  we  are  aware  the  only  experiments  with  similar  material  were 
conducted  by  Edsall  and  Miller  (see  bibliography)  on  infants  and  on 
man.  The  infants  digested  90  per  cent  or  over  of  the  nitrogen  in  the  bean 
periods  and  the  man  utilized  94  per  cent.  However,  the  bean  protein  fur- 
nished but  25  per  cent  of  that  of  the  infant's  food  and  only  about  12  per  cent 
of  the  protein  in  the  man's  dietary,  whereas  in  our  experiments  all  the  pro 
tein  was  supplied  by  the  bean  preparation. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  449 

TABLE  15. 


Crude  Bean  Protein  with  Agar  and  Bone  Ash. 


SUBJECT,  DOG  5 

Weight  at  beginning,  6.5  Kg. 
Weight  at  end,  6.2  Kg. 

PERIOD  X 

(4  days) 
Meat  Feeding 

PERIOD  XI* 

(3  days) 
Bean  Protein  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  2 
Bone  ash  5 
Water  100 

Estimated 

calories  520 

grams 

Bean  protein  300 
Sugar  20 
Lard  25 
Agar  2 
Bone  ash  5 
Water  (contain- 
ed in  the  bean 
protein)  240 
Estimated 

calories  560 

D&ily  .A.  vcrsi^cs 

4.65 
4.97 
5.23 
+0.26 

11.0 
0.32 
2.95 

93.8 

4.24 
5.08 
5.10 
+0.02 

39.3 
0.84 
2.15 

83.4 

*  Forced  feeding  necessary  throughout  the  period. 

TABLE  16. 

Crude  Bean  Protein  with  Agar  and  Bone  Ash. 

SUBJECT,  DOG  6 

Weight  at  beginning,  7.1  Kg. 
Weight  at  end,  6.8  Kg. 

PERIOD  XI 

(4  days) 
Meat  Feeding 

PERIOD  XII 

(3  days) 
Bean  Protein  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Meat  150 
Sugar  20 
Lard  20 
Agar  2 
Bone  ash  5 
Water  100 

Estimated 
calories  520 

grams 

Bean  protein  300 
Sugar  20 
Lard  25 
Agar  2 
Bone  ash  5 
Water  (contain- 
ed in  the  bean 
protein)  240 
Estimated 
calories  560 

Daily  Averages 

Daily  Averages 

3.84 
4.20 
5.23 
+1.03 

9.2 
0.36 
3.91 
93.1 

1 

3.62 
4.59 
5.10 
+0.51 

35.3 
0.97 
2.75 

81.0 

450  Utilization  of  Legume  Proteins 

TABLE  17. 


Crude  Bean  Protein  with  Agar  and  Bone  Ash. 


SUBJECT,  DOG  7 

Weight  at  beginning,  6.7  Kg. 
Weight  at  end,  6.4  Kg. 

PERIOD  X 

(4  days) 
Meat  Feeding 

PERIOD  XI 

(3  days) 
Bean  Protein  Feeding 

Composition  of  daily  diet  < 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Meat  150 

C?  mm****  -mm  OA 

sugar  ZD 
Lard  20 
Agar  2 
Bone  ash  5 
Water  100 

Estimated 

calories  520 

grams 

Bean  protein  300 
ougar  zU 
Lard  25 
Agar  2 
Bone  ash  5 
Water  (contain- 
ed in  the  bean 
protein)  240 
Estimated 

calories  560 

Daily  Averages 

Daily  Averages 

3.84 
4.16 
5.23 
+1.07 

10.0 
0.32 
3.20 

93.9 

3.51 
4.37 
5.10 
+0.73 

36.3 
0.86 
2.38 

83.1 

TABLE  18. 


Crude  Bean  Protein  with  Agar  and  Bone  As) 

i. 

SUBJECT,  DOG  5 

Weight  at  beginning,  6.2  Kg. 
Weight  at  end,  5.9  Kg. 

PERIOD  XII 

(4  days) 
Meat  Feeding 

PERIOD  XIII* 

(4  days) 
Bean  Protein 
Feeding 

PERIOD  XIV 

(4  days) 
Meat  Feeding 

Compostion    of  daily 
diet   ' 

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  pei 

grams 

Meat  100 

Sugar  25 
Lard  20 
Agar  2 
Bone  ash  5 
Water  150 

Estimated 
calories  450 

grams 

Bean  pro- 
tein 200 
Sugar  25 
Lard  25 
Agar  2 
Bone  ash  5 
Water  (160  gm. 
in  the  bean 
protein) 225 
Estimated 
calories  480 

grams 

Meat  100 

Sugar  25 
Lard  25 
Agar  2 
Bone  ash  5 
Water  150 

Estimated 
calories  450 

Daily  Averages 

Daily  Averages 

Dally  Averages 

3.15 
3.43 
3.49 
+0.06 

11  0 

0.28 
2.59 

91.8 

3.00 
3.54 
3.34 
-0.20 

26.5 
0.54 
2.05 

83.8 

2.64 
2.90 
3.28 
+0.38 

11.5 
0.26 
2.27 

92.1 

*  Forced  feeding  necessary  throughout  the  period. 


TABLE  19. 


Crude  Bean  Protein  with  Agar  and  Bone  Ash. 


SUBJECT,  DOG  6 

Weight  at  beginning,  6.6  Kg. 
Weight  at  end,  6.3  Kg. 

PERIOD  XIII 

(4  days) 
Meat  Feeding 

PERIOD  XIV 

(4  days) 
Bean  Protein 
Feeding 

PERIOD  XV 

(4  days) 
Meat  Feeding 

Composition    of  daily 
diet  I 

Nitrogen  output. 
Urine  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm.  . .  . 
Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  pei 
cent  

grams 

Meat  100 
Sugar  25 

T.nrH  90 

1  Jill < l  ZA) 

Agar  2 
Bone  ash  5 
Water  150 

Estimated 
calories  450 

grams 

Bean  pro- 
tein 200 
Sugar  25 

UnrH  9<i 

Agar  2 
Bone  ash  5 
Water  (160  gm. 
in  the  bean 
protein)  225 
Estimated 
calories  480 

grama 

Meat  100 

Sugar  25 
Lard  20 
Agar  2 
Bone  ash  5 
Water  150 

Estimated 
calories  450 

Daily  Averages 

Daily  Averages 

Dally  Averages 

2.56 
2.91 
3.49 
+0.58 

10.5 
0.35 
3.37 

89.8 

2.64 
3.33 
3.34 
+0.01 

28.3 
0.69 
2.45 

79.3 

2.40 
2.71 
3.28 
+0.57 

10.0 
0.31 
3.07 

90.6 

TABLE  20. 

Crude  Bean  Protein  with  Agar  and  Bone  Ash. 

SUBJECT,  DOG  7 

Weight  at  beginning,  6.4  Kg. 
Weight  at  end,  6.1  Kg. 

PERIOD  XII 

(4  days) 
Meat  Feeding 

PERIOD  XIII 

(4  days) 
Bean  Protein 
Feeding 

PERIOD  XIV 

(4  days) 
Meat  Feeding 

Composition    of    daily  \ 
diet  

Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per 
cent  

grams 

Meat  100 

Sugar  25 
Lard  20 
Agar  2 
Bone  ash  5 
Water  150 

Estimated 
calories  450 

grams 

Bean  pro- 
tein 200 
Sugar  25 
Lard  25 
Agar  2 
Bone  ash  5 
Water  (160  gm. 
in  the  bean 
protein)  225 
Estimated 
calories  480 

grams 

Meat  100 

Sugar  25 
Lard  20 
Agar  2 
Bone  ash  5 
Water  150 

Estimated 
calories  450 

Daily  Averages 

Daily  Averages 

Daily  Averages 

2.82 
3.05 
3.49 
+0.44 

8.5 

0.23 

2.74 

93.3 

2.70 
3.34 
3.34 
±0.00 

25.3 
0.64 
2.52 

80.9 

2.55 
2.81 
3.28 
+0.47 

9.5 

0.26 

2.70 

92.2 

45i 

452 


Utilization  of  Legume  Proteins 


TABLE  21. 

Crude  Bean  Protein  without  Agar  or  Bone  Ash. 


SUBJECT,  DOG  7 

Weight  at  beginning,  6.0  Kg. 
Weight  at  end,  5.9  Kg. 

PERIOD  XVI 

(4  days) 
Meat  Feeding 

PERIOD  XIX 

(3  days) 
Bean  Protein  Feeding 

Nitrogen  output. 

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per  cent  

grams 

Meat  100 
Sugar  25 
Lard  20 
Water  150 

Estimated 
calories  450 

grams 

Bean  protein  235 
Sugar  25 
Lard  20 
Water  (180  gm. 
in  the  bean 
protein)  230 
Estimated 
calories  480 

Daily  Averages 

Daily  Averages 

2.85 
2.95 
3.28 
+0.33 

1.5 
0.10 
7.06 
96.8 

2.58 
3.21 
3.43 
+0.22 

24.6 
0.63 
2.57 

81.5 

One  would  at  once  gain  the  impression  from  these  results  that 
the  legume  proteins  are  among  the  less  well  utilized  materials; 
nor  is  this  view  dispelled  when  we  attempt  to  analyze  certain  pos- 
sible contributing  causes.  The  bean  diets  contained  celluloses 
and  hemicelluloses,  substances  inherent  in  the  experimental  mate- 
rial and  entirely  or  for  the  most  part  indigestible.  The  influence 
upon  utilization  of  such  non-nitrogenous  matter  cannot  be  ascer- 
tained from  the  foregoing  tables.  In  a  few  instances  the  attempt 
has  been  made  to  determine  the  amounts  of  these  substances  which 
have  failed  to  disappear  from  the  alimentary  tract.  The  crude 
fiber  of  the  food  may  be  assumed  to  completely  reappear  in  the 
feces.1  The  undigested  hemicelluloses  of  the  excrement  was 
determined  by  the  method  outlined  in  a  previous  paper.2  These 
data  are  presented  in  Table  22  and  are  in  part  reproduced  in  Table 

1  Cf .  Swartz:  Transactions  of  the  Connecticut  Academy  of  Arts  and  Sci" 
ences,  xvi,  p.  268,  1911. 

2  Mendel  and  Fine:  This  Journal,  x,  p.  339,  1911. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  453 


23,  where  the  utilization  of  the  legume  proteins  is  compared  to 
that  of  the  protein  of  meat  diets  containing  comparable  or  greater 
amounts  of  indigestible  non-nitrogenous  matter.  From  the  latter 
table  it  is  apparent  that  the  presence  of  indigestible  non-nitrogenous 
matter  is  not  wholly  responsible  for  the  low  coefficients  of  diges- 
tibility of  the  proteins  of  the  soy  bean  and  crude  bean  prepara- 
tions, since  the  coefficients  for  the  nitrogen  of  meat  diets  including 
like  or  greater  amounts  of  such  substances  was  distinctly  higher. 
This  is  further  borne  out  in  the  following  two  experiments  (Tables 
24  and  25)  with  isolated  legume  proteins. 

table  22. 


Undigested  Carbohydrates  Derived  from  the  Food  Material.    Daily  Values. 


MATERIAL 

WEIGHT  OF 

COMPOSITION  OF  THE  FECES 

TABLE 

UNDER 

INGESTED 

INVESTIGATION 

LEGUME  (DRY) 

Crude  Fiber* 

Hemicellulose 

Total 

grams 

grams 

grams 

grams 

11 

Soy  Bean 

46 

1.0 

3.7 

4.7 

12 

Soy  Bean 

46 

1.0 

3.5 

4.5 

13 

Soy  Bean 

46 

1.0 

2.9 

3.9 

5 

Soy  Bean 

64 

1.5 

5.4 

6.9 

6 

Soy  Bean 

64 

1.5 

5.2 

6.7 

21 

CrudeBean 

Protein 

40 

4.4 

9.8 

14.2 

*Cf.  the  analyses  on  pp.  435  and  436. 


TABLE  23. 

Comparison  of  the  Utilization  of  Proteins  in  Relation  to  the  Content  of  Indi- 
gestible Non-Nitrogenous  Materials  in  the  Diet. 
Daily  Averages. 


TABLE 

NATURE  OF 
INGESTA 

NITROGEN  INTAKE 

INDIGESTIBLE 
NOls-NITROGENOUS 
MATTER  OF  THE 
DIET 

NITROGEN 
UTILIZATION 

grams 

grams 

per  cent 

11 

Soy  Bean 

3.3 

4.7 

80.2 

12 

Soy  Bean 

3.3 

4.5 

79.3 

13 

Soy  Bean 

3.3 

3.9 

83.8 

5 

Soy  Bean 

4.6 

6.9 

85.0 

6 

Soy  Bean 

4.6 

6.7 

84.0 

21 

Crude  Bean 

Protein 

3.2 

14.2 

81.5 

Meat* 

3.3 

13.0 

89.2 

Meat* 

3.3 

6.0 

91.0 

"These  data  will  be  discussed  more  fully  in  a  subsequent  paper  of  this  series.  In  each  case  the 
average  of  three  experiments  is  presented. 


454  Utilization  of  Legume  Proteins 

TABLE  24. 


Phaseolin  with  Agar  and  "Salts."* 


SUBJECT,  DOG  4. 

PERIOD  IX 

PERIOD  X 

PERIOD  XI 

Weight  at  beginning,  5.1  Kg. 

(3  days) 

(5  days) 

(5  days) 

Weight  at  end,  5.1  Kg. 

Meat  Feeding 

Phaseolin  Feeding 

Meat  Feeding 

grams 

grams 

grams 

Meat 

150 

Phaseolin 

38 

Meat 

150 

Sugar 

25 

Sugar 

25 

Sugar 

25 

Starch 

5 

Starch 

5 

Starch 

5 

Composition    of  daily 

Lard 

20 

Lard 

30 

Lard 

20 

diet   J 

Agar 

8 

Agar 

8 

Agar 

8 

Salts 

4 

Salts 

4 

Salts 

4 

Water 

200 

Water  300 

Water 

200 

Estimated 

Estimated 

Estimated 

calories  540 

calories  520 

calories 

540 

Nitrogen  output. 

Dally  Averages 

Daily  Averages 

Daily  Averages 

Urine  nitrogen,  gm  

4.45 

4.11 

4.28 

Total  nitrogen,  gm  

4.63 

5.32 

4.62 

Nitrogen  in  food,  gm  

5.27 

5.22 

5.20 

Nitrogen  balance,  gm. .  .  . 

+0.64 

-0.10 

+0.58 

Feces. 

Weight  air  dry,  gm  

9.4 

20.0 

12.4 

Nitrogen,  gm  

0.18 

1.21 

0.34 

Nitrogen,  per  cent  

1.93 

6.07 

2.77 

Nitrogen  utilization,  per 

96.5 

76.9 

93.4 

"The  ingredients  of  this  salt  mixture  were  reported  by  Mendel  and  Fine:  This  Journal,  x, 
p.  321,  1911. 


Phaseolin.  Dog,  Table  2  4:  The  food  mixture,  including  water, 
as  detailed  in  the  experimental  period  reported  in  Table  24,  was 
heated  on  a  water  bath  for  four  to  six  hours.  The  cooking  of  the 
food  mixture  resulted  in  the  gathering  together  of  the  material  in 
small  lumps,  which  may  possibly  account  for  the  very  unfavorable 
utilization.  We  do  not  believe  that  this  condition  appreciably 
influenced  the  result,  since  the  lumps  were  not  hard  and  solid,  but 
on  the  contrary,  quite  pervious.  Repeated  attempts  to  feed  this 
preparation  in  food  mixtures,  not  subjected  to  heat,  invariably 
resulted  in  complete  failure.  Ingestion  of  the  material  was  regu- 
larly followed  by  nausea  and  vomiting. 

Pea  Globulin.  Dog,  Table  25:  The  pea  globulin  as  used  in 
this  experiment  was  entirely  soluble  in  10  per  cent  sodium  chloride 
and  was  an  ideal  material  for  the  study  of  the  problem  in  hand. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  455 


The  food  mixture  was  not  heated  as  in  the  preceding  experiment, 
but  in  spite  of  this  the  material  gathered  together  in  small  pervious 
lumps,  when  the  water  was  added.  Although  the  utilization  does 
not  fall  far  below  that  of  meat,  fed  under  similar  conditions,  yet  in 
the  relatively  high  nitrogen  content  of  the  feces  we  note  the  evidence  of 
the  escape  of  a  portion  of  the  protein  from  digestion. 

As  far  as  we  are  aware,  Salkowski  is  the  only  other  worker  who 
has  studied  the  utilization  of  the  isolated  legume  protein.  This 
author  found  the  isolated  protein  of  the  horse  bean  to  be  94  per 
cent  utilized,  against  a  utilization  of  89  per  cent  for  the  untreated 
horse  bean.  It  should  be  noted  that  Salkowski's  protein  was  pre- 
pared by  extracting  the  beans  with  dilute  alkali  and  precipitating 
the  dissolved  protein  with  acid. 


TABLE  25. 

Pea  Globulin  with  Agar  and  Bone  Ash. 


SUBJECT  DOG,  6 

Weight  at  beginning,  4.9  Kg. 
Weight  at  end,  4.6  Kg. 


PERIOD  VI 

(4  days) 
Meat  Feeding 


PERIOD  VII 

(5  days) 
Pea  Globulin 
Feeding 


PERIOD  VIII 

(5  days) 
Meat  Feeding 


Meat 


grams 

150 


Composition    of  daily 
diet  


Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen,  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per 
cent  


Sugar 
Lard 
Agar 
Bone  Ash 


20 
20 
3 
7 


Water  100 
Estimated 
calories  520 


grams 

Pea  Glob- 
ulin 
Sugar 
Lard 
Agar 
Bone  Ash 
Water 


Meat 


grams 

150 


30 
20 
30 
3 
7 

200 


Estimated 
calories  470 


Sugar 
Lard 
Agar 
Bone  Ash 
Water 
Estimated 
calories  520 


20 
20 
3 
7 

100 


Daily  Averages 


Daily  Averages 


Daily  Averages 


4.13 
4.49 
4.93 
+0.45 

14.5 
0.35 
2.42 

92.9 


5.14 
5.70 
4.81 
-0.89 

15.6 
0.56 
3.62 


4.23 
4.62 
4.80 
+0.18 

15.0 
0.39 
2.59 

91.9 


Although  the  data  reported  in  this  paper  would  seem  to  warrant 
the  conclusion  that  the  leguminous  proteins  are  relatively  poorly 


456 


Utilization  of  Legume  Proteins 


utilized,  this  must  be  accepted  with  some  reservation,  at  least 
until  the  influence  of  varying  quantities  of  certain  indigestible 
materials  upon  the  utilization  of  easily  digested  substances  like 
meat  can  be  ascertained,  and  a  larger  number  of  trials  with  the 
isolated  proteins  are  carried  out. 

TABLE  26. 


Nitrogen  Balances  in  Soy  Bean  Experiment, 


SUBJECT 

TABLE 

SOY  BEAN 

MEAT 

Man  

1 

+0.21 

+1.67,  +1.06 

Dogl  

2 

-0.50 

-0.22,  +1.06 

Dog  5  

3 

+0.64 

+0.82,  +0.36 

Dog  7  

4 

+0.30 

+0.40,  +0.41 

Dog  5  

5 

-0.05 

+0.29 

Dog  6  

6 

+0.45 

+0.82 

Dog  7  

7 

+0.40 

+0.85 

Dog  5  

8 

+0.30 

+0.80 

Dog  6  

9 

+0.55 

+1.05 

Dog  7  

10 

+0.26 

+0.89 

Dog  5  

11 

-0.17 

+0.49 

Dog  6  

12 

+0.10 

+0.74 

Dog  7  

13 

-0.06 

+0.33 

TABLE  27. 

Nitrogen  Balances  in  Crude  Bean  Experiments. 

SUBJECT 

TABLE 

CRUDE  BEAN  PROTEIN 

MEAT 

Dog  5  

15 

+0.02 

+0.26 

Dog  6  

16 

+0.51 

+1.03 

Dog  7  

17 

+0.73 

+1.07 

Dog  5  

18 

-0.20 

+0.06,  +0.38 

Dog  6  

19 

+0.01 

+0.58,  +0.57 

Dog  7  

20 

+0.00 

+0.44,  +0.47 

Dog  7  

21 

+0.22 

+0.33 

TABLE  28. 


Nitrogen  Balances  in  Experiments  on  Phaseolin  and  Pea  Globulin, 


SUBJECT 

TABLE 

PHASEOLIN 

PEA  GLOBULIN 

MEAT 

Dog  4  ... 

24 

0 . 10 

+0.64,  +0.58 

Dog  6 

25 

-0.89 

+0.45,  +0.18 

Lafayette  B.  Mendel  and  Morris  S.  Fine  457 


In  Tables  26  to  28  are  collected  the  nitrogen  balances  obtained 
in  the  experiments  reported  in  the  present  paper.  The  soy  bean 
and  crude  bean  proteins  maintained  generous  positive  balances 
-  which  are,  however,  uniformly  smaller  than  those  for  meat.  This 
difference  is  explained  by  the  increased  elimination  of  fecal  nitrogen 
during  the  bean  periods.  A  similar  explanation  accounts  for  the 
negative  balance  during  the  phaseolin  period,  but  the  unfavorable 
nitrogen  balance  during  the  feeding  of  pea  globulin  is  presumably 
attributable  to  its  inherent  inadequacy. 

SUMMARY. 

In  comparison  with  the  other  vegetable  proteins  thus  far  reported 
in  this  series  of  studies,  the  legume  proteins  are  less  well  utilized. 
The  materials  investigated  principally  were  (1)  soy  bean  flour, 
free  from  starch ;  (2)  a  product  prepared  from  the  white  bean  by 
thoroughly  disintegrating  the  cells  and  dissolving  and  washing  out 
the  starch;  (3)  phaseolin — a  protein  isolated  from  the  white  bean; 
and  (4)  an  uncoagulated  globulin  from  the  garden  pea.  The  unfa- 
vorable results  with  the  soy  bean  and  white  bean  preparations  can 
be  explained  only  in  part  by  the  presence  of  cellulose  and  hemi- 
cellulose  in  these  products.  Such  considerations  cannot  be  applied 
to  the  data  for  phaseolin  and  pea  globulin. 

Attention  was  called  to  the  desirability  of  further  work  on  the 
isolated  legume  proteins,  and  on  the  influence  of  indigestible  non- 
nitrogenous  materials  upon  the  utilization  of  meat. 

The  observations  regarding  the  soy  bean  are  of  special  interest 
in  view  of  the  fact  that  this  product  has  lately  been  introduced 
quite  widely  as  an  adjuvant  to  the  dietary  of  diabetics. 

BIBLIOGRAPHY. 

Edsall  and  Miller:  American  Journal  of  the  Medical  Sciences,  cxxix, 
p.  663,  1905. 

Erismann:  Zeitschrift  fur  Biologic,  xlii,  p.  672,  1901. 
Hoffmann:  (Quoted  by  Voit :  Sitzungsberichte  der  Bayerische  Akademie, 
ii,  (4)  1869). 

Malfatti:  Wiener  Akademie,  Sitzungsberichte,  90  (iii),  p.  323,  1884. 
Oshima:  U.  S.  Department  of  Agriculture,  Office  of  Experiment  Stations, 
Bull.  159,  1905. 


458 


Utilization  of  Legume  Proteins 


Potthast:  Inaugural  Dissertation,  Leipzig,  1887. 
Prausnitz  :  Zeitschrift  fur  Biologie,  xxvi,  p.  228,  1890. 
Richter:  Archiv  fur  Hygiene,  xlvi,  p.  264,  1903. 
Rubner:  Zeitschrift  fiir  Biologie,  xvi,  p.  119,  1880. 
Salkowski:  Biochemische  Zeitschrift,  xix,  p.  83,  1909. 
Snyder:  Minnesota  Agricultural  Experiment  Station,  Bull.  92,  1905. 
Strumpell:  Deutsches  Archiv  fiir  klinische  Medizin,  xvii,  p.  108,  1875. 
Wait:  U.  S.  Department  of  Agriculture,  Office  of  Experiment  Stations, 
Bull.  187,  1907. 

Wintgen:  Veroffentlichungen  aus  d.  Gebiete  des  Militarsamtdtswesens, 
xxix,  p.  56,  1906. 

Woods  and  Mansfield  :  U.  S.  Department  of  Agriculture,  Office  of  Ex- 
periment Stations,  Bull.  149,  1904. 

Woroschilopf:  Berliner  klinische  Wochenschrift,  No.  8,  1873.  (Cited 
by  Strumpell.) 


Reprinted  from  The  Journal  of  Biological  Chemistry.  Vol.  XI,  No.  1,  1912 


STUDIES  IN  NUTRITION. 
V.    THE  UTILIZATION  OF  THE  PROTEINS  OF  COTTON  SEED. 

By  LAFAYETTE  B.  MENDEL  and  MORRIS  S.  FINE. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  September  25,  1911.) 

The  influence  of  cotton-seed  on  the  well-being  of  cattle  has  been 
extensively  investigated  in  this  country,  the  protein  of  this  mate- 
rial being  88  per  cent1  utilized  by  steers  or  sheep.  It  was  of  interest 
to  learn  to  what  extent  this  substance  was  utilized  by  dogs,  the 
alimentary  canal  of  which  more  closely  resembles  the  human  diges- 
tive tract.  Such  experiments  are  of  special  import,  inasmuch  as 
cotton-seed  flour  bids  fair  to  become  an  important  article  in  the 
human  dietary.  As  far  as  we  are  aware,  an  investigation  of  this 
nature  is  not  on  record.2 

EXPERIMENTAL  PART. 

Product  Employed. 

The  cotton-seed3  flour  of  these  experiments  was  a  deep  yellow 
impalpable  powder,  containing  7.4  per  cent  nitrogen.  Fraps4 
found  similar  samples  to  have  4.0  to  6.5  per  cent  crude  fiber. 
Cotton-seed  flour  contains  some  pentosans  but  no  starch.5 

1  Cf.  Fraps:  Texas  Agricultural  Experiment  Station,  Bull.  128,  1910. 

2  Correspondence  with  Dr.  C.  F.  Langworthy  and  Dr.  Marion  Dorsett, 
of  the  United  States  Department  of  Agriculture,  also  fail  to  reveal  any  lit- 
erature on  this  subject. 

3  Obtained  from  the  Southern  Cotton  Oil  Company,  Charlotte,  N.  C. 

4  Fraps:  loc.  cit. 
•  Fraps:  loc.  cit. 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY  XI,   NO.  I. 


I 


2 


Utilization  of  Cotton  Seed  Proteins 


Metabolism  Experiments. 

In  Table  1  are  recorded  three  experiments  on  the  utilization  of 
cotton-seed  flour.  The  usual  method  of  procedure6  prevailed. 
The  daily  supply  of  cotton-seed  contained  2  to  3  grams  of  crude 
fiber.  The  cotton-seed  feces  of  dogs  5  and  6  were  hydrolyzed 
according  to  the  method  outlined  in  a  previous  paper,7  and  yielded 
a  daily  average  of  respectively  5  and  3.5  grams  of  hemicelluloses. 
The  diets  of  these  two  dogs,  therefore,  included  7.5  and  6  grams  of 
indigestible  non-nitrogenous  substances.  This,  however,  cannot 
account  for  the  manifestly  poor  utilization  of  the  cotton-seed  nitro- 
gen.   The  coefficients  of  67  to  75  per  cent  for  cotton-seed  contrast 

TABLE  1. 


Cotton-seed  Flour. 


Dog  5 

Dog  6 

Dog  7 

PERIOD  XIX 

PERIOD  XX 

PERIOD  IX 

(4  days) 

(4  days) 

(3  days) 

Cotton-seed  Feed- 

Cotton-seed Feed- 

Cotton-seed Feed- 

ing 

ing 

ing 

grams 

grams 

grams 

Cotton-seed 

Cotton  seed 

Cotton-seed 

Flour  45 

Flour  45 

Flour  45 

Sugar  25 

Sugar  25 

Sugar  20 

Lard  20 

Lard  20 

Lard  25 

Composition  of  daily  diet  • 

Water  225 

Water  225 

Agar  3 

Bone  Ash  7 

Water  175 

Estimated 

Estimated 

Estimated 

calories  410 

calories  410 

calories  440 

Nitrogen  output. 

Daily  Averages 

Daily  Averages 

Daily  Averages 

Urine  nitrogen,  gm  

2.61 

2.61 

2.55 

3.51 

3.70 

3.45 

3.32 

3.32 

3.59 

Nitrogen  balance,  gm  

-0.20 

-0.38 

+0.14 

Feces. 

23.5 

23.9 

31.7 

Nitrogen,  gm  

0.91 

1.09 

0.90 

Nitrogen,  per  cent  

3.87 

4.57 

2.84 

Nitrogen    utilization,  pei 

cent  

72.6 

67.2 

74.9 

6  Cf.  Mendel  and  Fine:  This  Journal,  x,  p.  303,  1911. 
7Cf.  Mendel  and  Fine:  Ibid.,  x,  p.  339,  1911. 


Lafayette  B.  Mendel  and  Morris  S.  Fine 


3 


strikingly  with  those  of  88  to  93  per  cent  for  meat  diets  containing 
comparable  or  greater  amounts  of  such  indigestible  materials. 
(See  Table  2.)  There  is  of  course  the  possibility  that  the  cotton- 
seed flour  employed  in  this  study  contained  some  constituent8 
which  either  inhibited  secretion  or  promoted  premature  evacua- 
tion— conditions  which  would  result  in  poor  utilization. 

TABLE  2. 


Utilization  with  Reference  to  Indigestible  Materials  in  the  Diet* 
Daily  Averages. 


0 

o 

Q 

PERIOD 

DAYS 

NATURE  OF  INGESTA 

FIBER   CONTAINED  IN 
EXPERIMENTAL  MATE- 
RIAL OR  ADDED  TO  THE 
MEAT 

TOTAL  VOLUME  OF  IN- 
DIGESTIBLE MATERIAL 
IN  FOOD 

NITROGEN  INTAKE 

NITROGEN  UTILIZATION 

AVERAGE  NITROGEN 
UTILIZATION 

grams 

grams 

grams 

per  cent 

per  cent 

5 

xix 

4 

1     Cotton  seed  I 

3 

8t 

3.3 

72.6 

6 

XX 

4 

3 

6t 

3.3 

67.2 

71.6 

7 

ix 

3 

J  I 

3 

14$ 

3.G 

75.0 

5 

xviii 

4 

6 

6 

3.3 

90.5 

6 

xix 

4 

j    Meat  | 

6 

6 

3.3 

89.2 

91.0 

7 

xviii 

4 

6 

6 

3.3 

93.3 

5 

XV 

4 

1    Meat  f 

6 

13 

3.3 

91.6 

6 

xvi 

4 

>  Bone  ash,  5  grams  I 

6 

13 

3.3 

87.7 

89.2 

7 

XV 

4 

J    Agar,  2  grams  [ 

6 

13 

3.3 

88.3 

*  This  problem  will  be  treated  in  detail  in  a  subsequent  paper  of  this  series. 

t  Including  respectively  about  5  grams  and  3  grams  hemicelluloses  which  escaped  digestion. 

t  Including  approximately  4  grams  Indigestible  hemicelluloses  (exclusive  of  agar),  i.e.,  the  aver- 
age of  5  grams  and  3  grams.  An  actual  determination  of  the  hemicelluloses  of  the  feces  of  this 
experiment  was  not  made. 


8  Cf.  Crawford:  Journ.  of  Pharm.  and  Exper.  Ther.,  i,  No.  5,  p.  519,  1910. 


Reprinted  from  The  Journal  ok  Biological  Chrmistry,  Vol.  XI,  No.  I,  1912 


STUDIES  IN  NUTRITION. 

VI.    THE  UTILIZATION  OF  THE  PROTEINS  OF  EXTRACTIVE-FREE 
MEAT  POWDER;  AND  THE  ORIGIN  OF  FECAL  NITROGEN. 

By  LAFAYETTE  B.  MENDEL  and  MORRIS  S.  FINE. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  September  25,  1911.) 

CONTENTS. 


The  utilization  of  the  proteins  of  extractive-free  meat  powder   5 

Earlier  studies  with  this  and  related  materials   5 

Experimental  part   6 

Product  employed   6 

Metabolism  experiments   6 

On  the  origin  of  fecal  nitrogen   10 

Earlier  studies   11 

Experimental  part   15 

Influence  of  indigestible  non-nitrogenous  materials  upon  the 

nitrogen  statistics  of  the  feces  •.   15 

Influence  of  poorly  utilized  highly  nitrogenous  matter  upon 

the  nitrogen  statistics  of  the  feces   18 

Simultaneous  influence  of  both  types  of  materials  upon  the 

nitrogen  statistics  of  the  feces   19 

Effect  of  previous  thorough  evacuation  upon  utilization   21 

Estimation  of  "metabolic"  products  in  the  feces   21 

The  utilization  of  the  vegetable  proteins   23 


The  Utilization  of  Extractive-Free  Meat  Powder. 

earlier  studies. 

Forster  was  the  first  to  conduct  an  investigation  with  this  mate- 
rial. His  immediate  problem  was  the  question  of  salt  metabolism, 
but  incidentally  we  note  that  the  nitrogen  was  91  to  96  per  cent 
available.  During  the  past  twenty  years,  considerable  attention 
has  been  paid  to  the  comparative  utilization  of  fresh  meat  and 

S 


6 


The  Utilization  of  Proteins 


dried  meat  preparations,  for  example,  "soson,"  "somatose," 
"tropon,"  and  the  meat  residues  from  meat  extract  factories. 
Passing  over  the  literature  previous  to  1901,  we  may  dwell  briefly 
upon  the  results  obtained  by  Prausnitz,  which  are  in  general  accord 
with  those  of  the  earlier  workers.  The  average  coefficient  of 
digestibility  of  dried  meat  was  90  per  cent  against  a  coefficient 
of  93  per  cent  for  fresh  meat.  Moreover  the  nitrogen  concentra- 
tion of  the  dried-meat-feces  was  1.35  to  1.76  per  cent  higher  than 
the  fresh  meat  feces.  These  facts  make  it  probable  that  a  portion 
of  the  dried  meat  had  escaped  absorption.  Prausnitz  also  showed 
that  dried  meat  was  less  readily  digested  in  artificial  gastric  juice 
than  fresh  meat.  He  accounted  for  these  phenomena  on  the 
assumption  that  a  not  inappreciable  length  of  time  elapses  before 
the  dried  meat  particles  are  sufficiently  "hydrated"  to  permit  the 
digestive  enzymes  to  operate.  Max  Voit  found  similar  although 
less  striking  differences. 

Considerable  work  has  also  been  accomplished  with  dried  blood 
preparations,  but  a  consideration  of  these  investigations  would 
lead  us  too  far  afield. 

EXPERIMENTAL  PART. 

Product  Employed. 

The  meat  residue1  employed  in  the  present  studies  was  a  light 
brown  impalpable  powder,  containing  13.2  per  cent  of  nitrogen, 
8.9  per  cent  of  ether  extract,  2.5  per  cent  of  ash,  and  7.0  per  cent  of 

moisture. 

Metabolism  Experiments. 

Tables  1-3.  During  these  experiments,  the  methods  described 
in  a  previous  paper2  were  followed.  The  utilization  of  the  nitrogen 
of  meat  powder  is  distinctly,  although  slightly  lower  than  that  of  fresh 
meat.  The  relatively  high  nitrogen  concentration  of  the  meat  powder 
feces  is  indicative  of  a  loss  of  this  material  through  the  excrement. 
These  points  are  concisely  presented  in  the  accompanying  brief 
tabular  summary. 

1  Obtained  from  Armour  and  Company. 

2  Mendel  and  Fine:    This  Journal,  x,  p.  303,  1911. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  7 

Summary  of  the  Data  on  Nitrogen  Utilization  (see  Tables  1-8) 


MEAT  POWDER 

FRESH  MEAT  (AVERAGES) 

DOG 

Nitrogen 
utilization 

Nitrogen  in  feces 

Nitrogen 
utilization 

Nitrogen  in  feces 

1 

4 
4 

per  cent 

91.3 
89.3 
91.0 

per  cent 

2.98 
3.81 
3.87 

per  cent 

94.0 
94.5 
93.7 

per  cent 

1.94 
2.04 
2.36 

TABLE  1. 

Extract-free  Meat  Powder. 


SUBJECT,  DOG  1 

Weight  at  beginning,  14.6  kg. 
Weight  at  end,  14.6  kg. 


period  v 
(4  days) 
Meet  Feeding 


PERIOD  VI* 

(5  days) 
Meat  Powder 
Feeding 


PERIOD  VII 

(4  days) 
Meat  Feeding 


Composition  of  daily  diet  I 


Nitrogen  output. 

Urine  nitrogen,  gm  

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization,  per 
cent  


grams 

Meat  300 

Lard  60 
Agar  5 
Bone  ash  15 
Water  300 
Estimated 
calories  1070 


grams 


Meat  Pow 
der 
Lard 
Agar 
Bone  Ash 
Water 


80 
60 
5 
15 
500 


Daily  Averages 


8.81 
9.38 
10.44 
+1.06 

29.5 
0.57 
1.95 

94.5 


Estimated 
calories  860 


Daily  Averages 


8.76 
9.68 
10.53 
+0.85 

31.0 
0.92 
2.98 

91.3 


Meat 


grams 

300 
60 


Lard 
Agar  5 
Bone  Ash  15 
Water  300 
Estimated 
calories  1070 


Daily  Averages 


8.47 
9.15 
10.46 
+1.31 

35.2 
0.68 
1.92 

93.5 


*  Food  almost  entirely  forced. 


8  The  Utilization  of  Proteins 

TABLE  2. 


Extract-free  Meat  Powder. 


SUBJECT,  DOG  4 

Weight  at  beginning  4.9  kg. 
Weight  at  end,  5.1  kg. 

PERIOD  V 

(4  days) 
Meat  Feeding 

PERIOD  VI 

(5  days) 
Meat  Powder 
Feeding 

PERIOD  VII 

(4  days) 
Meat  Feeding 

Composition  of  daily  diet.  < 

Nitrogen  output. 

Total  nitrogen,  gm  

Nitrogen  in  food,  gm  

Nitrogen  balance,  gm  

Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen    utilization,  per 
cent  

grams 

Meat  loU 

Sugar  25 
Starch  5 
Lard  20 
Bone  Ash  10 
Water  200 

Estimated 
calories  570 

grams 

Meat 

Powder  39 
Sugar  25 
Starch  5 
Lard  25 
Agar  8 
"Salts"  4 
Water  260 
Estimated 
calories  510 

grams 

Meat  150 

Sugar  25 
Starch  5 
Lard  20 
Agar  8 
"Salts"  4 
Water  200 
Estimated 
calories  570 

Daily  Averages 

Daily  Averages 

Daily  Averages 

4.50 
5.40 
+0.90 

15.0 
0.27 
1.79 

95.0 

o .  yo 
4.50 
5.13 
+0.63 

14.4 
0.55 
3.81 

89.3 

4.70 

5.40 
+0.70 

14.0 
0.32 
2.29 

94.1 

Lafayette  B.  Mendel  and  Morris  S.  Fine 


TABLE  3. 
Extract-free  Meat  Powder. 


SUBJECT,  DOG  4 

Weight  at  beginning,  5.1  kg. 
Weight  at  end,  5.2  kg. 


Composition  of  daily  diet. 


Nitrogen  output. 
Urine  nitrogen,  gm 

Total  nitrogen,  gm  

Nitrogen  in  food,  gm. . . 
Nitrogen  balance,  gm. . 
Feces. 

Weight  air  dry,  gm  

Nitrogen,  gm  

Nitrogen,  per  cent  

Nitrogen  utilization, 
cent  


per 


PERIOD  XI 

(5  days) 
Meat  Feeding 


4.28 
4.62 
5.20 
+0.58 

12.4 
0.34 
2.77 

93.4 


PERIOD  XII 

(5  days) 
Meat  Powder 
Feeding 


grams 

grams 

grams 

Meat 

150 

Meat 
Powder 

40 

Meat 

150 

Sugar 

25 

Sugar 

25 

Sugar 

25 

Starch 

5 

Starch 

5 

Starch 

5 

Lard 

20 

Lard 

25 

Lard 

20 

Agar 

8 

Agar 

8 

Agar 

4 

"Salts" 

4 

"Salts" 

4 

Bone  Ash 

8 

Water 

200 

Water 

300 

Water 

200 

Estimated 

Estimated 

Estimated 

calories 

570 

calories 

510 

calories 

570 

Daily  Averages 

Daily  Averages 

Daily  Averages 

4.51 
4.98 
5.26 
+0.28 

12.3 
0.47 
3.87 

91.0 


PERIOD  XIII 

(4  days) 
Meat  Feeding 


4.46 
4.77 
5.22 
+0.45 

16.0 
0.31 
1.96 

94.0 


IO 


The  Utilization  of  Proteins 


On  the  Origin  of  Fecal  Nitrogen. 

In  previous  papers3  of  this  series  we  have  followed  the  current 
custom  of  basing  the  data  for  nitrogen  utilization  upon  the  rela- 
tion of  the  nitrogen  appearing  in  the  excrement  to  that  of  the 
ingesta.  This  procedure  would  be  strictly  correct  only  in  case  the 
fecal  nitrogen  consisted  entirely  of  food  residues.  As  a  matter  of 
fact,  there  is  abundance  of  evidence  in  the  literature  to  demon- 
strate that  fecal  nitrogen  in  great  part  emanates  from  "  metabolic 
products."4  Obviously  an  adequate  understanding  of  the  source 
of  fecal  nitrogen  and  the  conditions  influencing  its  excretion  is 
essential  for  the  proper  interpretation  of  experiments  on  nitrogen 
utilization.  In  the  earlier  papers  referred  to  we  have  at  times 
pointed  out  that  an  apparently  poor  utilization  was  probably  in- 
duced by  the  indigestible  matter — cellulose,  hemicellulose — inher- 
ent in  the  experimental  material.  The  influence  of  such  materials 
upon  utilization  has  not  always  been  fully  appreciated.  Rubner, 
and  later  Wicke,  did  indeed  call  attention  to  the  unfavorable 
effect  of  cellulose  upon  the  utilization  of  bread  nitrogen;  but  in 
these  cases  it  is  difficult  to  decide  in  what  measure  the  insufficiently 
ruptured  cells  are  responsible  for  the  low  coefficients  of  digesti- 
bility, and  to  what  extent  the  latter  is  to  be  attributed  to  the  cel- 
lulose per  se.  This  question  is  not  satisfactorily  answered  by  the 
poor  utilization  of  meat  obtained  by  Hoffmann  when  coarsely  cut 
straw  was  added  to  the  diet.  Such  coarse  particles  probably 
unduly  irritated  the  digestive  tract,  resulting  in  increased  secre- 
tion and  peristalsis.  Lothrop  demonstrated  an  increased  elimi- 
nation of  fecal  nitrogen  when  bone  ash  was  added  to  the  diet. 

In  the  present  paper  the  nitrogen  of  the  excrement  under  a 
variety  of  conditions  is  discussed  briefly  from  the  historical  aspect;5 
data  purporting  to  show  to  what  extent  indigestible  non-nitroge- 
nous substances  may  influence  the  amount  and  character  of  the 
feces  are  presented;  and  a  plan  of  experimentation  is  proposed, 

3  See  footnotes  20-24,  pp.  23  and  24. 

4  By  this  term  is  understood  intestinal  secretions,  cast  off  cells,  bacteria, 
etc.  For  a  consideration  of  the  important  role  of  bacteria  in  this  respect 
and  the  literature  related  thereto,  see  MacNeal,  Latzer  and  Kerr:  Journ. 
of  Infect.  Dis.,  vi,  p.  123,  1909. 

5  For  a  more  detailed  review  reference  is  made  to  Tsuboi  (see  bibli- 
ography). 


Lafayette  B.  Mendel  and  Morris  S.  Fine  n 


with  which  it  seems  possible  to  approximately  determine  to  what 
degree  the  nitrogen  excreted  in  the  feces  is  derived  from  undigested 
or  indigestible  nitrogenous  constituents  of  the  ingesta.  Were 
this  known,  the  term  "  utilization"  would  be  eminently  appropriate. 

EARLIER  STUDIES. 

Feces  in  Starvation. 

Man.  The  accompanying  table  presents  oft  quoted  data6 
obtained  from  the  professional  fasters,  Cetti  and  Breithaupt,  and 
from  certain  patients. 

Daily  Nitrogen  Excreted  through  the  Feces  in  Starvation. 


gram 

Cetti  .   0.32 

Breithaupt   0.12 

Patient   (stenosis   of  oesophagus)   0.45 

Neurasthenic   0.22 

Neurasthenic   0.17 

Average   0.26 


Dogs.  Bidder  and  Schmidt,  and  Voit  early  observed  that  during 
starvation  black  pitch-like  feces  were  obtained  from  dogs.  The 
latter  obtained  daily  2  grams  of  feces  (=  0.15  gram  of  nitrogen) 
from  a  dog  of  30  kilos.  The  studies  of  Miiller  offer  further  illus- 
trative data. 


Daily  Feces  Obtained  from  Starving  Dogs  (Miiller,  1884). 


BODY  WEIGHT 

FECES  WEIGHT 
DRY 

FECAL  NITROGEN 

FECAL  NITROGEN 
PER  KILO  BODY 
WEIGHT 

kilo 

grams 

per  cent 

grams 

gram 

43 

4.8 

5.0 

0.24 

0.005G 

30 

2.4 

8.0 

0.19 

0.0063 

30 

1.4 

8.0 

0.11 

0.0037 

23 

2.8 

5.3 

0.15 

0.0065 

7 

0.7 

7.5 

0.05 

0.0071 

0.0058 

6  Taken  from  Schmidt  and  Strasburger  (see  bibliography),  p.  115. 


The  Utilization  of  Proteins 


Benedict  has  pointed  out  that  the  amount  of  feces  formed  during 
starvation  is  probably  much  smaller  than  is  indicated  by  earlier 
studies.  Fasting  feces  are  in  great  part  derived  from  retained 
fecal  matter,  resulting  from  the  food  immediately  preceding  the 
period  of  inanition.  This  is  owing  to  diminished  peristalsis  con- 
sequent upon  the  withdrawal  of  food. 

With  Nitrogen-Free  Diets. 
The  accompanying  table  embodies  results  obtained  by  Rieder. 


Nitrogen  Eliminated  through  Feces  on  Nitrogen-free  Diet  (Rieder). 


SUBJECT 

FECES 
WEIGHT 
DRY 

FECAL  NITROGEN 

FOOD 

grams 

per  cent 

gram 

Man  

13.4 

4.08 

0.54 

485  grams  cakes  of  starch,  sugar 

and  fat. 

Man  

15.4 

5.69 

0.87 

159  grams  cakes  of  starch,  sugar 

and  fat. 

Man  

13.4 

5.85 

0.78 

147  grams  cakes  of  starch,  sugar 

and  fat. 

Dog  

3.0 

3.67 

0.11 

70  grams  starch. 

Dog  

6.0 

3.85 

0.22 

140  grams  starch. 

Rubner  (1879)  reported  similar  results.  Tsuboi  fed  dogs  for 
periods  of  six  to  nine  days  on  cakes  made  of  starch,  fat  and  sugar, 
and  obtained  data,  which  are  in  accord  with  the  above. 


Nitrogen  Eliminated  through  Feces  on  Nitrogen-free  Diet  (Tsuboi). 


FECES 
WEIGHT  DRY 

FECAL  NITROGEN 

FOOD 

Starch 

Sugar 

Fat 

grams 

per  cent 

gram 

grams 

grams 

grams 

2.6 

5.1 

0.14 

0 

0 

0 

5.8 

4.1 

0.24 

70 

12 

50 

12.9 

4.4 

0.57 

200 

25 

80 

There  can  of  course  be  no  question  as  to  the  source  of  fecal 
nitrogen  in  the  above  experiments. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  13 


With  Meat  Diets. 

The  most  interesting  work  bearing  upon  the  nitrogen  of  the  feces 
obtained  with  meat  diets  and  the  relation  of  the  amount  of  meat 
ingested  to  the  nitrogen  thus  eliminated  was  contributed  by  Muller. 


Influence  of  Meat  Diet  on  Fecal  Nitrogen  in  Dogs  (30-35  kilos)  (Muller). 


MEAT 

FECES  WEIGHT 
DRY 

FECAL  NITROGEN 

NITROGEN  UTILIZED 

grams 

grams 

per  cent 

gram 

per  cent 

0 

2.0 

7.96 

0.15 

500 

5.1 

6.50 

0.30 

98.2 

1000 

9.2 

6.50 

0.55 

9.8.4 

1500 

10.2 

6.50 

0.67 

98.7 

1800 

10.3 

6.50 

0.70 

98.9 

2000 

11.1 

6.50 

0.80 

98.8 

2500 

15.4 

6.50 

1.00 

98.8 

It  is  clear  from  this  summary  that  the  nitrogen  of  the  feces  does 
not  increase  in  proportion  to  the  amount  of  meat  eaten. 

That  the  fecal  nitrogen  incident  to  a  meat  diet  is  essentially  of 
metabolic  origin,7  is  very  convincingly  brought  out  by  Fritz 
Voit.  After  a  loop  of  the  intestine  had  been  isolated,  a  dog  was 
fed  with  meat.  It  was  found  that  the  contents  of  the  loop  resem- 
bled the  feces  in  appearance  and  nitrogen  content.  Moreover, 
when  calculated  to  unit  surface  the  absolute  amount  of  dry  sub- 
stance in  the  loop  compared  favorably  with  that  of  the  feces. 
Equally  significant  is  the  recent  study  of  Mosenthal,  who  also 
worked  with  isolated  intestinal  loops.  This  author  estimated  that 
the  succus  entericus  contained  nitrogen  equivalent  to  35  per  cent 
of  the  nitrogen  ingested,  and  300  to  400  per  cent  of  the  nitrogen 
of  the  feces.  Nitrogen  equivalent  to  at  least  25  per  cent  of  that 
of  the  intake  must  therefore  have  been  reabsorbed. 

From  the  foregoing  there  can  be  no  doubt  that  the  feces  resulting 
from  a  thoroughly  digestible  food  such  as  meat  are  almost  solely 
of  "metabolic  origin."  Prausnitz  has  attempted  to  give  this  more 
widespread  application. 

7  By  an  ingenious  microscopical  method,  Kermauner  (see  bibliography) 
showed  that  in  man  but  one  per  cent  or  less  of  the  ingested  meat  reappeared 
in  the  feces. 


j  4  The  Utilization  of  Proteins 


Composition  of  Feces  on  Various  Diets  (Prausnitz). 


FECES — DRY 

NUMBER 

PERSON 

MAIN  FOOD 

Nitrogen 

Ether 
Extract 

Ash 



per  cent 

per  cent 

per  ccTit 

1 

H. 

Rice 

8.83 

12  A 

15.4 

2 

H. 

Meat 

8.75 

16.0 

14.7 

3 

M. 

Rice 

8. 37 

18.2 

11.0 

4 

M. 

Meat 

9. 16 

16.0 

12.2 

5 

W.P. 

Rice 

8.59 

15.9 

12.6 

6 

W.P. 

Meat 

8.48 

17.5 

13.1 

7 

J.Pa. 

Rice 

8.25 

14.5 

8 

J.Pa. 

Meat 

8.16 

15.2 

9 

F.Pi. 

Rice 

8.70 

16.1 

10 

F.Pi. 

Meat 

9.05 

15.1 

11 

d.Cl. 
(vegetarian) 

Rice 

8.78 

18.6 

12.0 

Average 

8.65 

16.4 

13.8 

12 

M. 

Mixed  diet 

6.76 

25.3 

12.0 

13 

H. 

Mixed  diet 

6.63 

25.8 

14.9 

14 

H. 

Mixed  diet 

6.07 

30.1 

15.0 

The  excreta  from  the  above  diets  (Nos.  1  to  11)  contained  no 
starch,  and  the  composition  of  the  feces  did  not  alter  materially 
as  the  character  of  the  food  changed.  Such  feces  Prausnitz  con- 
sidered "normal  feces. "  When,  however,  the  food  contains  mate- 
rial of  a  less  digestible  nature,  the  composition  may  change. 
Where  this  indigestible  material  is  cellulose  the  nitrogen  content 
of  the  feces  is  lowered  (Nos.  12  to  14);  if  a  nitrogenous  substance, 
the  nitrogen  content  might  be  expected  to  be  raised. 

Schierbeck  recognizes  three  types  of  individuals:  (1)  those  that 
consistently  have  feces  with  low  nitrogen  concentration  (about  4 
per  cent)  whatever  the  nature  of  the  diet  may  be;  (2)  those  that 
under  these  conditions  have  feces  of  high  nitrogen  percentage 
(6-7  per  cent) ;  and  (3)  those  in  whom  coarse  food  yields  feces  of 
low  nitrogen  percentage,  and  readily  absorbed  material  produces 
feces  with  nitrogen  concentration  as  high  as  8  per  cent. 

We  are  inclined  to  agree  with  Benedict  that  during  starvation 
the  formation  of  feces  is  reduced  to  a  practically  negligible  quan- 
tity. When  a  material  such  as  meat  is  eaten  whose  protein 
utilization,  estimated  according  to  the  usual  custom,  is  at  least  95 


Lafayette  B.  Mendel  and  Morris  S.  Fine 


'5 


per  cent,  the  resulting  feces  are  for  the  most  part  of  metabolic 
origin.  It  has  been  shown  that  the  feces  from  such  a  diet  represent 
a  very  small  portion  of  the  originally  secreted  intestinal  juice,  the  latter 
having  been  absorbed  in  great  part  before  reaching  therectum.  Ob- 
viously the  degree  to  which  this  secretion  is  reabsorbed  will  depend 
upon  the  rate  of  peristalsis,  which  in  turn  is  influenced  by  the  mass  and 
character  of  material  in  the  intestine.  Hence,  if  to  a  meat  diet  an 
indigestible  or  less  digestible  material  is  added,  thus  stimulating 
peristalsis,  more  metabolic  products*  must  escape  reabsorption  If 
we  deal  with  a  non-nitrogenous  material,  e.g.,  agar,  bone  ash  or  crude 
fiber,  the  percentage  nitrogen  of  the  feces  will  of  course  be  lower. 
If  the  comparatively  indigestible  material  is  highly  nitrogenous  like 
protein,  the  nitrogen  concentration  will  be  higher;  and  if  both  types  of 
indigestible  materials  are  present,  the  percentage  of  nitrogen  may  be 
indistinguishable  from  that  found  in  meat-feces.  Illustrative  data 
follow. 

EXPERIMENTAL  PART. 

The  conduct  of  these  experiments  did  not  differ  essentially  from 
that  of  trials  described  in  previous  papers.  The  quantities  of 
meat  and  indigestible  non-nitrogenous  materials  can  be  learned 
from  the  tables;  the  amounts  of  water,  sugar  and  lard  approxi- 
mated those  employed  in  previous  experiments. 

The  influence  of  indigestible  non-nitrogenous  materials  upon  the 
nitrogen  statistics  of  the  feces  is  illustrated  in  Tables  4  and  5.  In 
Table  4  the  contrast  is  made  between  feces  resulting  from  meat  and 
feces  accruing  from  an  identical  diet  to  which  3  grams  of  agar 
plus  7  grams  of  bone  ash  had  been  added  daily.  In  Table  5  a 
similar  contrast  is  drawn  between  meat-  and  meat-crude-fiber 
feces.  The  data  are  briefly  summarized  in  Table  6.  The  increase 
in  absolute  fecal  nitrogen  due  to  the  addition  of  indigestible 
materials  to  the  diet  is  manifest,  although  the  nitrogen  intake  did 
not  vary.  Thus  the  fecal  nitrogen  of  (1)  is  increased  60  per  cent 
by  the  addition  of  10  grams  of  indigestible  non-nitrogenous  sub- 
stances, and  that  of  (3)  is  augmented  133,  133,  and  192  per  cent9 

8  Possibly  also  food  residues  and  products  of  digestion. 

9  Too  great  a  quantitative  significance  should  not  be  placed  upon  these 
figures,  as  an  accurate  isolation  of  pure  meat-feces  is  almost  impossible 
even  when  special  precautions  are  taken. 


i6 


The  Utilization  of  Proteins 


TABLE  4. 

Influence  of  Agar  +  Bone  Ash  upon  the  Feces  Resulting  from  a  Meat  Diet. 
Daily  Averages. 


NATURE  OF  INGEST A 


+3  >> 


XX 

xxviii 
i 

iii 
iv 
viii 


/  Meat,  sugar,  lard  =  4.6  \ 
\     to  4.9  gm.  nitrogen  j 


As  above + 


Agar  3  gm. 
Bone  Ash 
7  gm 


Average  of  1  and  2. 
Average  of  3  to  6. . . 


grams 

4.5 
3.4 

13.2 
14.5 
15.5 
15.0 

4.0 

14.5 


gram 

0.22 
0.16 

0.29 
0.36 
0.40 
0.35 

0.19 
0.37 


per  cent 

4.95 
4.62 


xxi 
xxix 


iv 
vi 
viii 


Meat,  etc.,  as  for  Dog  5 


Meat,  etc.,  with  indiges- 
tible materials,  as  for 
Dog  5 


Average  of  7  and  8. 
Average  of  9  to  12. . 


3.5 
4.4 

12.2 
13.6 
14.5 
15.0 

4-0 
13.8 


0.22 
0.24 

0.28 
0.36 
0.35 
0.39 

0.23 
0.34 


xx 

xxviii 
i 

iii 
v 

vii 


Meat,  etc.,  as  for  Dog  5 


Meat,  etc.,  with  indiges- 
tible materials,  as  for 
Dog  5 


Average  of  13  and  14- 
Average  of  15  to  18. . . 


3.2 
3.8 

12.5 
12.8 
12.7 
12.8 

3.5 
12.7 


0.19 
0.15 

0.28 
0.23 
0.26 
0.24 

0.17 
0.25 


Lafayette  B.  Mendel  and  Morris  S.  Fine  17 


TABLE  5. 


Influence  of  Crude  Fiber  upon  the  Feces  Resulting  from  a  Meat  Diet. 
Daily  Averages. 


xvn 
xix 


xvi 
xviii 


Xll 


xiv 
xv 


xv 
xvi 


xiv 
xv 


NATURE  OF  INGESTA 


Meat,  etc.,  =  3.3  gm. 

nitrogen 
As  above  +  6  gm. 

crude  fiber* 

Meat,  etc.,  as  for  Dog  5 
Meat,  etc.,  +  6  gm.  crude 
fiber  as  for  Dog  5 

Meat,  etc. ,  as  for  Dog  5 
Meat,    etc.,    +    6  gm. 
crude  fiber  as  for  Dog  5 

Average  of  1,3,5  , 

Average  of  2,  4,6  


Meat,  etc.,  +  2  gm.  agar  + 

5  gm.  bone  ash 
The  same 

The  same  +  6  gm.  filter 
paper 

Meat,  etc.,  +  2  gm.  agar 

+  5  gm.  bone  ash 
The  same 

The  same  +  6  gm.  filter 
paper 

Meat,  etc.,  +  2  gm.  agar 

+5  gm.  bone  ash 
The  same 

The  same  +  6  gm.  filter 
paper 

Average  of  7,  8,  10,  11,  13,  14 
Average  of  9, 12, 15  


0.4f 

10.1 
1.9 

10.0 
1.5 

8.5 

1.7 
9.5 


11.0 
11.5 

17.7 

10.5 
10.0 

18.0 

8.5 
9.5 


0.02f 

0.30 
0.13 

0.34 
0.10 

0.21 

0.12 
0.28 


0.28 
0.26 

0.27 

0.35 
0.31 

0.40 

0.23 
0.26 


18.0 

0 

38 

2 

14 

88.3 

10.2 

0 

28 

2 

79 

91.6 

17.9 

0 

35 

1 

97 

89.2 

per  cent 

5.32 

2.97 
7.03 

3.52 
7.06 

2.51 

6.4' 
3.00 


2.59 
2.27 

1.55 

3.37 
3.07 

2.23 

2.74 
2.70 


w  « 

u  S3 

£  3 


per  cent 

99.4J 

90.5 
96.0 

89.2 
96.8 

93.3 

96.4 
91.0 


91.8 
92.1 

91.6 

89.8 
90.6 

87.7 

93.3 
92.2 


*  Newspaper  (0.1  per  cent  nitrogen)  was  thoroughly  disintegrated  under  water, 
t  These  values  are  abnormally  low  owing  to  poor  separation  of  feces  of  successive  periods. 
They  are  not  included  in  the  averages, 
t  Omitted  from  the  averages. 

THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  XI  NO.  1. 


i8 


The  Utilization  of  Proteins 


TABLE  6. 

The  Influence  of  Indigestible  Non-Nitrogenous  Materials  upon  the  Nitrogen 
Statistics  of  Meat-Feces.    (Summary  of  Tables  4  and  5) .    Daily  Averages. 


DUMBER 

NUMBER  OF  EXPERI- 
MENTS AVERAGED 

TAKE 

UME  INDI- 
ION-NITRO- 
ATERI  AL 
fHE  MEAT 

FECES 

'ILIZATION 

REFERENCE  J 

NITROGEN  IN 

TOTAL  V  O  L 1 
GESTIBLE  I 
GENOUS  M 
ADDED  TO  1 

Weight 
(Air  Dry) 

Nitrogen 

Nitrogen 

NITROGEN  Ul 

grams 

grams 

grams 

gram 

per  cent 

per  cent 

1 

6* 

4.6 

0 

3.8 

0.20 

5.2 

95.7 

2 

12** 

4.6 

10 

13.7 

0.32 

2.3 

92.7 

3 

3t 

3.3 

0 

1.7 

0.12 

6.5 

96.4 

4 

3| 

3.3 

6 

9.5 

0.28 

3.0 

91.0 

5 

6§ 

3.3 

7 

10.2 

0.28 

2.8 

91.6 

6 

3|| 

3.3 

13 

17.9 

0.35 

2.0 

89.2 

•  Cf.  Table  4,  Nos.  1,  2,  7,  8,  13,  14. 
**  Cf.  Table  4,  Nos.  3  to  6,  9  to  12,  15  to  18. 

t  Cf.  Table  5,  Nos.  1,3,5. 
k  |  Cf.  Table  5,  Nos.  2,  4,  6. 

§  Cf.  Table  5,  Nos.  7,  8,  10, 11, 13, 14. 

||  Cf.  Table  5,  Nos.  9,  12,  15. 

by  the  addition  to  the  diet  of  6,  7,  and  13  grams  respectively  of 
such  materials.  The  low  nitrogen  concentration  of  the  feces  of 
(2),  (4),  (5),  and  (6)  is  characteristic  of  diets  of  thoroughly  util- 
ized materials  including  much  indigestible  non-nitrogenous  matter. 
The  nitrogen  concentration,  however,  is  not  sufficiently  diminished 
to  compensate  for  the  increased  volume  of  feces — hence  the  above 
increment  in  absolute  fecal  nitrogen  and  the  correspondingly  low- 
ered coefficients  of  digestion. 

Illustrations  of  the  influence  of  poorly  utilized  highly  nitrogenous 
matter  upon  the  nitrogen  statistics  of  the  feces  are  especially  con- 
spicuous in  certain  data  already  published10  and  which  are  repro- 
duced in  Table  7.  The  nitrogen  concentration  of  the  phaseolin- 
feces  is  6.1  per  cent  against  2.3  per  cent  for  that  of  feces  resulting 
from  a  meat  diet  fed  under  conditions  identical  with  those  attend- 
ing the  phaseolin  feeding.    A  similar  though  less  striking  example 

10  Mendel  and  Fine:  This  Journal,  x,  p.  433,  1912:  Table  24  (phaseolin); 
Table  25  (pea  globulin);  Tables  10-11  (soy  bean). 


Lafayette  B.  Mendel  and  Morris  S.  Fine  19 


is  offered  in  the  case  of  the  pea  globulin  experiment.  Nos.  5  to 
8  of  this  table  disclose  how  closely  the  nitrogen  concentration  of  feces 
accruing  from  diets  containing  both  poorly  utilized  highly  nitrogenous 
materials  and  indigestible  non-nitrogenous  materials  may  simulate 
the  corresponding  value  for  meat  feces. 


TABLE  7. 

The  Influence  of  Poorly  Utilized  Highly  Nitrogenous  Materials  upon  the 
Nitrogen  Statistics  of  the   Feces.    Daily  Averages. 


REFERENCE 
NUMBER 

NATURE  OF  INGESTA 

NITROGEN 
INTAKE 

Weight 
(Air  Dry) 

FECES 

Nitrogen 

Nitrogen 

NITROGEN 
UTILIZA- 
TION 

grams 

grams 

grams 

per  cent 

per  cent 

1 

Phaseolin 

5.2 

20.0 

1.21 

6.1 

76.9 

2 

Meat* 

5.2 

10.9 

0.26 

2.3 

95.0 

3 

Pea  Globulin 

4.8 

1,5.6 

0.56 

3.6 

88.3 

4 

Meatf 

4.8 

14.7 

0.37 

2.5 

92.4 

5 

Soy  Bean 

4.6 

15.9 

0.57 

3.6 

87.6 

6 

Meat 

4.6 

3.8 

0.15 

3.8 

96.9 

7 

Soy  Bean 

3.3 

14.0 

0.65 

4.7 

80.2 

8 

Moot, 

3.3 

0.4| 

0.02| 

5.3 

99.4 

*  Average  of  fore  and  after  periods, 
t  Average  of  fore  and  after  periods. 
X  See  second  note  to  Table  5,  this  paper. 


Obviously  the  fecal  nitrogen  concentration  by  itself  is  not  a  safe 
criterion11  by  which  to  judge  the  digestibility  of  a  material.  The 
nitrogen  of  the  voluminous  meat-cellulose-feces  may  be  almost 
entirely  of  metabolic  origin  and  yet  be  present  in  relatively  low 
concentration;  whereas  a  soy  bean  diet  may  yield  feces  composed 
in  great  part  of  highly  nitrogenous  undigested  food  residues,  the 
nitrogen  concentration,12  however,  being  comparable  to  that  of 
meat-feces. 

11  Tsuboi  (see  bibliography),  p.  80,  likewise  believes  that  one  should  be 
conservative  in  drawing  conclusions  from  this  one  factor. 

12  Tsuboi  (loc.  cit.,  p.  81),  has  made  a  similar  statement.  He  points  out 
that  in  Rubner's  studies,  peas  were  poorly  utilized  (72  per  cent)  and  yet 
the  nitrogen  concentration  of  the  feces  was  7.3  per  cent,  thus  according 
closely  with  that  of  6.9  per  cent  for  the  nitrogen  concentration  of  feces  from 
meat  which  was  97  per  cent  utilized. 


20 


The  Utilization  of  Proteins 


Benedict  has  called  attention  to  the  difficulty  encountered  in 
satisfactorily  isolating  feces  accruing  from  a  particular  diet,  owing 
to  the  lagging  behind  of  fecal  material  from  the  preceding  diet. 
Our  own  experience  testifies  to  this  difficulty.  It  was  especially 
pronounced  where  the  experimental,  preceding  and  succeeding 


TABLE  8. 

Influence  of  Thorough  Evacuation  upon  Nitrogen  Statistics  of  Feces. 
Daily  Averages. 


FECES 

b5 

NUMBE 

o 
o 
a 

DAYS 

PERIOD 

NATURE  OF  INGE8TA 

Weight 
Air  Dry 

Nitrogen 

Nitrogen 

NITROGE 
UTILIZATI 

grams 

gram 

per  cent 

per  cent 

1 

5 

4 

xxiii 

Meat,  etc.,  +  10  gm.  Agar 
(  =  4.6  gm.  Nitrogen 

15.7 

0.52 

3.33 

88.6 

2 

5 

3 

xxiv 

Meat,  etc.,  +  10  gm.  Bone 
Ash 

13.8 

0.26 

1.90 

94.3 

3 

5 

4 

XXV 

Meat,  etc.,  +  10  gm.  Agar 

13.5 

0.40 

2.95 

91.4 

4 

6 

4 

xxiv 

Meat,  etc.,  +  10  gm.  Agar 

15.5 

0.53 

3.42 

88.5 

5 

6 

3 

XXV 

Meat,  etc.,  +  10  gm.  Bone 
Ash 

15.2 

0.32 

2.12 

93:1 

6 

6 

4 

xxvi 

Meat,  etc.,  +  10  gm.  Agar 

12.8 

0.37 

2.92 

92.0 

7 

7 

4 

xxiii 

Meat,  etc.,  +  10  gm.  Agar 

14.8 

0.45 

3.02 

90.3 

8 

7 

3 

xxiv 

Meat,  etc.,  +  10  gm.  Bone 
Ash 

14.0 

0.28 

1.99 

94.0 

9 

7 

4 

XXV 

Meat,  etc.,  +  10  gm.  Agar 

11.9 

0.32 

2.70 

93.1 

Average  of  1,  4,7  

15.3 

0.50 

3.26 

89.1 

A  verage  of  2,5,  8  

14.3 

12.7 

0.29 

2.00 

93.8 

Average  of  3,6,9  

0.36 

2.86 

92.2 

Lafayette  B.  Mendel  and  Morris  S.  Fine 


21 


diets  were  all  composed  of  thoroughly  digested  materials  and  the 
resulting  feces  were  not  adequate  stimuli  to  peristalsis.  This 
difficulty  was  obviated  in  a  measure  when  the  experimental  period 
was  preceded  and  succeeded  by  a  2-3  day  period  of  a  meat  diet 
including  10  grams  of  bone  ash  daily. 

This  lag  and  the  effect  of  previous  thorough  evacuation  upon  util- 
ization is  illustrated  in  Table  8.  The  first  period  for  each  dog 
(Nos.  1,  4,  7)  was  preceded  by  a  period  of  wheat  gluten,  which  is 
very  well  utilized.13  After  thorough  evacuation,  it  is  clear  (Nos.  3, 
6,  9)  that  the  apparent  utilization  is  considerably  improved. 

Estimation  of  " Metabolic11  Products  in  the  Feces. 

Investigators  have  sought  a  method  whereby  the  prominent 
part  taken  by  alimentary  waste  products  in  the  formation  of  feces 
could  be  determined  with  some  degree  of  accuracy.  This  would 
enable  one  to  estimate  what  proportion  of  the  feces  is  due  to  undi- 
gested food  residues.  Processes  have  been  proposed  which  involve 
treating  the  feces  with  pepsin-HCl  or  dilute  alkali.  Data  thus 
obtained  are  of  doubtful  value.  Equally  unsatisfactory  are  those 
procedures  which  involve  subtracting  from  the  experimental  feces 
the  equivalent  of  fecal  material  obtained  during  starvation  or  on  a 
thoroughly  digested  non-nitrogenous  diet.  The  plan  generally 
followed  in  the  present  work,  namely  the  comparison  of  experimen- 
tal feces  with  feces  obtained  from  a  control  meat  diet  is  likewise  not 
always  free  from  objection.  None  of  the  above  methods  take 
into  account  the  influence  of  undigested  masses  upon  the  degree 
of  reabsorption  of  the  intestinal  juice.  We  propose  the  following 
plan14  which  seems  to  avoid  most  of  the  above  shortcomings: 

1.  Determine  the  volume  and  nitrogen  of  feces  resulting  from 
the  material  under  investigation. 

2.  Determine  the  fecal  nitrogen  resulting  from  a  nitrogen-free 
diet  to  which  has  been  added  an  amount  of  indigestible  non- 

13  Cf.  Mendel  and  Fine:  This  Journal,  x,  p.  324,  1911. 

14  Tsuboi  has  applied  a  similar  principle  to  certain  results  reported  by 
Rubner.  The  nitrogen  eliminated  on  a  starch  diet  was  subtracted  from  that 
excreted  in  feces  of  comparable  volume  resulting  from  diets  of  wheat  bread 
and  maccaroni.  The  food  nitrogen  actually  escaping  utilization  could 
thus  be  computed. 


22 


The  Utilization  of  Proteins 


nitrogenous  matter15  that  will  yield  approximately  the  same  vol- 
ume of  feces  as  was  obtained  in  (l). 

3.  Subtract  the  fecal  nitrogen  of  (2)  from  that  of  (1).  This 
excess  of  nitrogen  is  presumably  due  to  undigested  or  unabsorbed 
nitrogenous  matter  of  the  food  material. 

An  experiment  with  a  nitrogen-free  diet  including  indigestible 
non-nitrogenous  matter  follows: 

A  6  kilo  bitch  was  fed  for  four  days  on  a  mixture  of  35  grams  of  sugar,  45 
grams  of  lard,  200  grams  of  water  and  10  grams  of  agar.  On  this  diet  13.2 
grams  of  feces  with  a  nitrogen  concentration  of  2.44  per  cent  were  obtained 
daily.  There  were  thus  eliminated  through  the  feces  0.32  gram  of  nitrogen 
daily,  which  was  obviously  of  metabolic  origin.  This  result  makes  it  prob- 
able that  the  feces  from  meat  diets,  containing  similar  amounts  of  indiges- 
tible non-nitrogenous  matter,  (see  Table  6)  are  likewise  made  up  entirely 
of  alimentary  waste — proof  in  itself  that  meat  nitrogen  is  100  per  cent  utilized. 

From  the  single  experiment  above  reported  and  from  Table  6, 
No.  6,  we  may  conclude  that  a  thoroughly  digested  material  may 
yield  13.2  to  17.9  grams  of  feces  and  yet  the  nitrogen  (0.32-0.35 
grams)  thus  eliminated  will  be  of  "metabolic"  origin.  Hence  in 
feces  of  comparable  volumes16  all  nitrogen  in  excess  of  0.32-0.35 
gram  may  be  attributed  to  the  nitrogen  of  the  food.  This  prin- 
ciple is  applied  in  Table  9. 

"Utilization,  "as  the  term  is  employed  in  the  last  column  of  this 
table,  exactly  expresses  our  meaning.  The  actual  utilization  of 
soy  bean  nitrogen17  is  90.3-92.8  per  cent  and  that  for  the  crude 
bean  protein  is  91.8  per  cent.  If  anything  the  latter  value  is 
low,  as  24.6  grams  of  meat-feces  would  probably  contain  more 
than  0.35  gram  of  nitrogen. 

15  The  choice  of  indigestible  adjuvant  is  a  matter  of  some  moment,  as  these 
materials  may  vary  in  their  ability  to  stimulate  peristalsis. 

16  This  of  course  applies  only  for  dogs  of  approximately  the  same  weight 
(5  to  7  kilos.)  as  those  in  these  experiments. 

17  Soy  bean  is  reported  (Wolff -Lehmann :  Landw.  Fiitterungslehre,  cited 
by  Schulze  und  Castoro:  Zeitschr.  f.  physiol.  Chem.,  xli,  p.  455,  1904)  as 
having  10  per  cent  of  its  nitrogen  present  as  non-protein.  The  latter  may  be 
more  thoroughly  utilized  than  the  protein  constituents,  and  thus  the  utili- 
zation calculated  for  the  total  nitrogen  intake  would  be  greater  than  is 
actually  the  case  for  the  soy  bean  protein.  Excepting  the  soy  bean  and 
cotton-seed  flours,  the  preparation  of  the  materials  employed  in  this  series 
of  studies  renders  contamination  with  nitrogenous  non-protein  matter 
unlikely. 


Lafayette  B.  Mendel  and  Morris  S.  Fine  23 


The  Utilization  of  the  Vegetable  Proteins. 

About  the  thorough  utilization  of  the  proteins  of  wheat18  there 
is  no  question.  The  probability  that  those  of  barley19  and  corn20 
are  equally  available  was  pointed  out  in  previous  papers  of  this 
series.  With  regard  to  the  legume  proteins21  we  must  for  the 
present  conclude  that  the  presence  of  indigestible  non-nitrogenous 
materials  camiot  entirely  account  for  their  low  coefficients  of  diges- 


TABLE  9. 

Utilization  as  Estimated  from  the  Portion  of  Fecal  Nitrogen  Derived  from  Food 
Residues.    Daily  Averages. 


NUMBER  OP  EX- 
PERIMENTS 
AVERAGED 

NATURE  OF  INGESTA 

NITROGEN 
INTAKE 

FECES 

NITROGEN  FROM 
UNDIGESTED 
FOOD 

NITROGEN  UTILIZATION 

Weight 
(Air  Dry) 

Nitrogen 

As 

Ordinarily 
Estimated 

Actual 
Utilization 

gram 

grams 

gram 

gram 

per  cent 

per  cent 

1 

Nitrogen-free  diet 

including    10  gm. 

agar 

0.0 

13.2 

0.32 

0.00 

3 

Meat  diet* 

3.3 

17.9 

0.35 

0.00 

89.2 

100.0 

6 

Soy  beanf 

4.6 

17.2 

0.68 

0.33 

85.3 

92.8 

3 

Soy  bean| 

3.3 

13.0 

0.64 

0.32 

81.1 

90.3 

1 

Crude  bean  protein§ 

3.4 

24.6 

0.63 

0.28 

81.5 

91.8 

*  Cf .  Table  6,  No.  6  this  paper. 

t  Cf.  Mendel  and  Fine:   This  Journal,  x,  p.  433,  1912.   Tables  5-10. 
t  Cf.  Mendel  and  Fine:   loc.  cit.   Tables  11  to  13. 
§  Cf.  Mendel  and  Fine:  loc.  cit.   Table  21. 


tibility.  These  proteins  appear  to  be  less  readily  affected  by  the 
digestive  processes  than  those  of  barley  or  corn.  This  resistance 
is  even  more  pronounced  in  the  case  of  the  cotton-seed  protein.22 
Nevertheless,  future  research  with  the  isolated  proteins  may  modify 
our  opinion  with  regard  to  these  two  last  classes  of  materials. 

The  lack  of  animal  extractives  in  vegetable  materials  has  at 
times  been  thought  to  be  the  cause  of  the  apparently  poor  utili- 
zation of  plant  foods  in  comparison  with  those  of  animal  origin. 

18  Cf.  Mendel  and  Fine:  This  Journal,  x,  p.  303,  1911. 

19  Cf.  Mendel  and  Fine:  Ibid,  x,  p.  339,  1911. 

20  Cf.  Mendel  and  Fine:  Ibid.,  x,  p.  345,  1911. 

21  Cf.  Mendel  and  Fine:  Ibid,  x,  p.  433,  1912. 

22  Cf.  Mendel  and  Fine:  Ibid,  xi,  p.  1,  1912. 


24 


The  Utilization  of  Proteins 


The  evidence  bearing  upon  this  seems  to  be  inconclusive.  Bischoff 
showed  that  meat  extracts  did  not  appreciably  influence  the  diges- 
tibility of  bread.  Thompson  came  to  the  opposite  conclusion. 
Effront  noted  that  meat  extracts  exert  a  favorable  influence  upon 
the  availability  of  vetegable  diets,  but  this  could  not  be  confirmed 
by  Wintgen.  The  fact  that  the  proteins  of  wheat,  and  probably 
those  of  barley  and  corn  also,  are  thoroughly  utilized  lends  support 
to  the  view  that  the  secretory  influences  of  the  extractive  materials 
play  a  minor  role  in  the  ultimate  utilization.  It  was  pointed  out  in 
an  earlier  paper  that  certain  wheat  preparations  evoked  intense 
nausea  in  man,  and  necessitated  forced  feeding  in  the  dog  experi- 
ments, but  were,  nevertheless,  thoroughly  digested.  This  would 
suggest  that  psychic  secretion  does  not  influence  the  ultimate 
utilization  to  any  great  extent.23  It  would  be  interesting  to  study 
the  relation  of  gastric  secretion  to  ultimate  utilization  by  means 
of  a  sequestered  stomach. 

Studies  on  the  digestibility  of  vegetable  proteins  in  vitro  are  not 
lacking.  Rothe24  found  that  in  24  out  of  26  of  his  experiments,  the 
coefficients  of  digestibility  were  above  90  per  cent.  The  coefficients 
for  the  legumes  averaged  about  95  per  cent.  In  these  studies,  2 
grams  of  material  were  acted  upon  by  250  cc.  of  concentrated  gas- 
tric juice  for  forty-eight  hours.  As  yet,  it  is  uncertain  to  what 
extent  in  vitro  experiments  of  this  kind  can  be  held  comparable  to 
studies  on  the  utilization  of  proteins  from  the  alimentary  tract. 
It  may  be  noted  that  artificial  digests  are  not  contaminated  with 
"  metabolic"  products,  and  this  may  explain  the  high  coefficients 
of  digestibility  obtained  in  such  trials  compared  with  those  result- 
ing from  experiments  in  vivo.  Studies  on  animals  where  this  factor 
has  been  taken  into  account  yield  results  not  very  unlike  those 
obtained  in  artificial  digestion  experiments  (see  Table  9) . 

A  study  of  the  literature  on  the  availability  of  vegetable  materials 
reveals  an  interesting  situation.  In  instances  where  the  protein 
has  been  very  poorly  utilized,  the  carbohydrate,  on  the  contrary, 
has  rarely  been  less  than  95  per  cent  digested.  This  is  illustrated 
in  the  accompanying  table,  containing  data  gathered  at  random. 

23  Cf.  Schmidt  und  Strasburger:  "Die  Fazes  des  Menschen,"  p.  17,  Berlin 
1910.  Also  Osborne  and  Mendel:  Carnegie  Institution  of  Washington, 
Publication  No.  156,  p.  5,  1911. 

24  Rothe:  Zeitschr.f.  physiol.  Chem.,  li,  p.  185, 1907,  (contains  the  literature). 


Lafayette  B.  Mendel  and  Morris  S.  Fine  25 


Table  Illustrating  the  Simultaneous  Poor  Utilization  of  Protein  and  Good 
Utilization  of  Carbohydrate. 


NATURE  OF  FOOD 

Kidney  beans  

White  beans  

Cow  peas  

Rice,  barley,  and  vegetables 

Rice  and  barley  

Cooked  rice  

Barley  


COEFFICIENTS  OF  DIGESTIBILITY 

Protein 

Carbohydrate 

per  cent 

per  cent 

77 

94 

78 

96 

70 

87 

76 

97 

67 

99 

76 

99 

60 

97 

AUTHOR  OR 
COMPILER 


Wait25 
Wait 
Wait 
Oshima26 
Oshima 
Oshima 
Oshima 


It  is  possible  that  the  starch  has  been  completely  utilized  and 
the  carbohydrate  of  the  feces  is  in  reality  hemicellulose. 


BIBLIOGRAPHY. 


THE  UTILIZATION  OF  MEAT  POWDERS  AND  ALLIED  MATERIALS. 

Abderhalden  und  Ruehl:  Zeitschrift  fur  physiologische  Chemie,  lxix, 
p.  301,  1910. 

Ellinger:  Zeitschrift  fur  Biologie,  xxxiii,  p.  190,  1896. 
Forster:  Ibid.,  ix,  1873.  (cited  by  Atwater  and  Langworthy:  U.  S. 
Dept.  of  Agriculture,  Office  of  Experiment  Stations,  Bull.  45,  1897. 
Hildebrand:  Zeitschrift  fur  physiologische  Chemie,  xviii,  p.  180,  1894. 
Imabuchi:  Ibid.,  lxiv,  p.  1,  1910. 
Maas  :  Medizinische  Klinik,  No.  8,  1906. 

Muller:  Miinchener  medizinische  Wochenschrift,  xlvii  (2),  p.  1769,  1900. 
Neumann:  Ibid.,  xlv  (1),  p.  72,  1898  and  xlvi  (l),  p.  42,  1899. 
Neumeister:  Deutsche  medizinische  Wochenschrift,  xix,  pp.  866  and  1169, 
1893. 

Plauth  :  Zeitschrift  fur  diatetische  und  physikalische  Therapie,  i,  p.  62, 1898. 
Prausnitz:  Zeitschrift  fur  Biologie,  xlii,  p.  377,  1901. 
Salkowski:  Biochemische  Zeitschrift,  xix,  p.  83,  1909. 
Schmilinsky  und  Kleine:  Miinchener  medizinische  Wochenschrift,  xlv 
(2),  p.  995,  1898. 
Strauss:  Therapeutische  Monatshefte,  xii,  p.  241,  1898. 
Voit:  Zeitschrift  filr  Biologie,  xlv,  p.  79,  1904. 


26  Wait:  U.  S.  Dept.  of  Agriculture,  Office  of  Experiment  Stations,  Bull. 
187,  p.  53,  1907. 
26  Oshima:  Ibid.,  Bull.  159,  1905. 


26 


The  Utilization  of  Proteins 


ORIGIN  OF  FECAL  NITROGEN  AND  CONDITIONS  INFLUENCING  ITS  EXCRETION. 

Benedict:  " The  Influence  of  Inanition  on  Metabolism,"  p.  347,  Carnegie 
Institution  of  Washington,  1907. 

Bidder  und  Schmidt:  "Die  Verdauungssdfte  und  der  Stoffwechsel," 
1852. 

Bischoff:  Zeitschrift  fur  Biologie,  v,  p.  454,  1869. 

Effront:  Fiinfter  internationeller  Kongress  fiir  angewandte  Chemie,  p. 
97,  1904.    (Cited  by  Wintgen.) 

Hoffmann:  (Cited  by  Voit:  Sitzungsberichte  der  bayerischen  Akademie, 
ii,  (4),  1869). 

Kermauner:  Zeitschrift  fur  Biologie,  xxxv,  p.  316,  1897. 
Lothrop:  American  Journal  of  Physiology,  xxiv,  p.  297,  1909. 
Mosenthal:  Journal  of  Experimental  Medicine,  xiii,  p.  319,  1911. 
Muller:  Zeitschrift  fur  Biologie,  xx,  p.  326,  1884.    (Cited  by  Tsuboi.) 
Prausnitz:  Ibid.,  xxxv,  p.  335,  1897. 
Rieder:  Ibid.,  xx,  p.  378,  1884.    (Cited  by  Tsuboi.) 
Rubner:   Ibid.,  xv,  p.  115,  1879;  also  Ibid.,  xix,  p.  45,  1883. 
Schmidt  und  Strasburger:  "Die  Faeces  des  Menschen,"  p.  115,  Berlin, 
1903. 

Schierbeck:  Archiv  fur  Hygiene,  li,  p.  62,  1904. 
Thompson:  Zentralblatt  fur  Physiologie,  No.  17,  p.  814,  1910. 
Tsuboi:  Zeitschrift  fur  Biologie,  xxxv,  p.  68,  1897. 
Voit  (C):  Ibid.,  ii,  p.  308,  1866.    (Cited  by  Tsuboi.) 
Voit  (P.):  Ibid.,  xxix,  p.  325,  1893.    (Cited  by  Tsuboi.) 
Wicke:  Archiv  fiir  Hygiene,  xl,  p.  349,  1890. 

Wintgen:  Veroffentlichungen  aus  d.  Gebiete  des  Militdrsanitdtswesens, 
xxix,  p.  56,  1906. 


Reprinted  from  Tiik  Journal  of  Biological  Chemistry,  Vol.  X,  No.  2,  1911 


STUDIES  IN  CARBOHYDRATE  METABOLISM. 

I.    THE  INFLUENCE  OF  HYDRAZINE  UPON  THE  ORGANISM,  WITH 
SPECIAL  REFERENCE  TO  THE  BLOOD  SUGAR  CONTENT.1 

PLATE  III. 

By  FRANK  P.  UNDERHILL. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  July  28,  1911.) 

The  constancy  of  the  sugar  content  of  the  blood  under  normal 
conditions  constitutes  one  of  fundamental  axioms  of  physiology. 
It  has  been  universally  assumed  that  a  certain  supply  of  sugar  in 
the  blood  is  essential  to  normal  metabolic  rhythm  and  that  even 
under  distorted  physiological  conditions,  as  in  inanition,  the  organ- 
ism is  capable  of  furnistiing  the  requisite  materials  from  its  own 
economy  for  the  maintenance  of  the  blood  sugar  constant.      f  , 

Attempts  to  upset  this  nice  adjustment  have  resulted  in  the  main 
in  the  temporary  establishment  of  an  excessive  quantity  of  sugar 
in  the  blood.  Thus  hyperglycaemia  may  follow  the  introduction 
of  various  drugs  into  the  body  or  may  be  produced  by  the  induc- 
tion of  different  pathological  states.  On  the  other  hand,  with 
the  exception  of  phloridzin  and  uranium  glycosurias,  little  has 
been  known  concerning  the  conditions  necessary  to  diminish  the 
content  of  blood  sugar.  ; 

The  practical  significance  of  the  investigations  upon  changes  in 
blood  sugar  content  obviously  lies  in  the  importance  which  the 
results  obtained  from  such  research  may  bear  to  the  interpreta- 
tion and  treatment  of  human  diabetes.  It  has  been  assumed  that 
if  the  abnormal  state  which  exists  in  diabetes  could  be  experimen- 
tally reproduced,  some  hope  for  the  prevention  or  alleviation  of 
the  condition  might  be  realized.    Such,  however,  has  not  been 

XI  am  much  indebted  to  Dr.  M.  S.  Fine  for  aid  in  carrying  out  some  of 
these  experiments. 

1 5Q 


160      Hydrazine  and  Carbohydrate  Metabolism 


the  case  for,  with  the  possible  exception  of  pancreatic  diabetes, 
an  exact  experimental  duplication  of  the  conditions  existing  in 
human  diabetes  has  not  yet  been  made. 

Recent  investigations  in  the  production  of  hypoglycaemia, 
notably  those  of  Frank  and  Isaac,1  with  phosphorus,  suggest  a 
possible  control  of  the  blood  sugar  content  which  may  lead  to  a 
distinct  advance  in  our  knowledge  concerning  certain  phases  of 
carbohydrate  metabolism. 

The  present  paper  is  the  first  of  a  series  in  which  it  is  planned 
to  present  various  aspects  of  carbohydrate  metabolism  under 
conditions  in  which  the  sugar  of  the  blood  is  experimentally  dimin- 
ished. The  method  employed  for  the  production  of  this  condi- 
tion was  the  administration  of  the  diamine,  hydrazine. 

THE  INFLUENCE  OF  HYDRAZINE  UPON  THE  ORGANISM  IN  GENERAL. 

In  their  paper  on  "The  Influence  of  Hydrazine  upon  Inter- 
mediary Metabolism  in  the  Dog,"  Underhill  and  Kleiner2  have 
described  the  general  effects  of  hydrazine  upon  the  organism  in 
the  following  words: 

"The  researches  of  Borissow,3  of  Pohl4  and  of  Poduschka6  have  demonstrat- 
ed the  relatively  great  toxicity  of  this  compound  and  have  defined  the 
series  of  manifestations  following  its  introduction  into  the  body.  With 
doses  of  0.1  gram  hydrazine  sulphate  per  kilo  of  body  weight  subcutaneously 
injected  vomiting  is  observed  which  is  succeeded  by  extreme  restlessness. 
There  is  augmentation  of  the  heart  beat  which  later  falls  below  the  normal 
and  respiratory  difficulty  is  accompanied  by  general  paralysis.  At  this 
stage  a  short  period  of  coma  usually  ensues  which  terminates  in  death.  The 
entire  cycle  of  events  is  completed  within  a  very  few  days.  Coincident  with 
the  symptoms  noted  above  is  the  appearance  in  the  urine  of  varying  quan- 
tities of  protein  and  bile  pigments  " 

From  his  histological  study  of  the  action  of  hydrazine,  Wells6 
concluded  that 

"hydrazine  seems  to  be  a  poison  with  an  almost  specific  effect  upon  the 
cytoplasm  of  the  parenchymatous  cells  of  the  liver,  for  when  the  poison  is 

1  Frank  and  Isaac:  Arch.  f.  exp.  Path.  u.  Pharmakol.,  lxiv,  p.  274,  1911. 

*  Underhill  and  Kleiner:    This  Journal,  iv,  p.  165,  1908. 

'Borissow:  Zeitschr.  f.  physiol.  Chem.,  xix,  p.  499,  1894. 

4 Pohl:  Arch.  f.  exp.  Path.  u.  Pharmakol,  xlviii,  p.  367,  1902. 

6 Poduschka:  Ibid.,  xliv,  p.  59,  1900. 

•Wells:  Journ.  of  Exp.  Med.,  x,  p.  457,  1908. 


Frank  P.  Underhill 


161 


given  subcutaneously  this  tissue  alone  shows  evident  structural  alterations, 
although  equal  or  greater  amounts  must  reach  other  organs  or  tissues.  It 
seems  to  have  remarkably  little  effect  upon  other  than  hepatic  cells,  and 
does  not  cause  any  appreciable  destruction  of  red  corpuscles;  slight  hemor- 
rhages are  occasionally  produced,  but  much  less  than  by  other  poisons  with 
a  similar  effect  upon  the  liver.  It  attacks  only  the  cytoplasm  of  the  liver 
cells,  never  affecting  the  nucleus  primarily,  and  causes  a  profound  fatty 
metamorphosis  of  the  type  commonly  referred  to  as  "fatty  degeneration." 
In  this  respect  it  resembles  phosphorus,  from  which  it  differs  in  two  impor- 
tant particulars.  Hydrazine  attacks  first  the  cells  in  the  center  of  the 
lobules,  while  phosphorus  shows  its  first  and  most  marked  effects  upon  the 
peripheral  cells;  and  secondly,  phosphorus  usually  causes  marked  fatty 
changes  in  the  myocardium,  the  kidneys,  and  indeed  throughout  the  body, 
whereas  the  effects  of  hydrazine  seem  to  be  limited  almost  absolutely  to 
the  liver.  The  unknown  poisons  of  acute  yellow  atrophy  and  eclampsia, 
and  most  of  the  bacterial  poisons,  attack  first  and  chiefly  the  nuclei  of  the 
liver  cells,  in  contrast  to  the  strictly  cytoplasmic  effects  of  hydrazine.  Phos- 
phorus also  effects  the  nuclei  more  than  does  hydrazine.  On  this  account 
the  recovery  of  the  liver  to  normal  after  hydrazine  poisoning  is  remark- 
ably rapid  and  complete,  there  being  no  permanent  anatomical  alterations 
after  recovery  from  a  most  severe  non-fatal  poisoning." 

Despite  the  grave  injury  to  the  liver  it  has  been  demonstrated 
that  "the  most  striking  feature  of  the  action  of  hydrazine  upon 
the  animal  body  is  the  absence  of  abnormal  relationships  in  the 
principal  urinary  constituents"1  and  the  presence  of  abnormal 
substances,  such  as  lactic,  oxybutyric  and  diacetic  acids,  acetone 
or  reducing  bodies  could  not  be  detected  in  the  urine,  although  in 
one  instance  there  was  a  separation  of  a  small  quantity  of  cystine.2 

Subsequent  experience  with  hydrazine  has  afforded  additional 
facts  concerning  the  general  influence  of  this  poison  upon  the  body 
of  the  dog  which  are  of  considerable  importance  for  the  conduct 
of  future  investigation.  In  the  first  place  the  subcutaneous  admin- 
istration of  hydrazine  sulphate  in  the  dosage  of  50  mgms.  per 
kilo  presents  an  entirely  different  series  of  symptoms  than  a  simi- 
lar introduction  of  100  mgms.  per  kilo  body  weight.  In  both 
instances  there  is  the  same  initial  picture,  i.e.,  vomiting  and  ex- 
treme restlessness.  With  the  smaller  dosage,  however,  the  ani- 
mal appears  merely  drowsy  and  stupid  during  the  first  day.  In 
general  upon  the  second  day  the  dog  seems  practically  normal 
with  the  noteworthy  exception  that  there  may  be  evidence  of 

1  Underhill  and  Kleiner:  loc.  ext. 
2 Underhill  and  Kleiner:  loc.  ext. 


1 62      Hydrazine  and  Carbohydrate  Metabolism 


extreme  weakness  especially  noticeable  in  the  hind  limbs.  Food 
is  refused.  The  animals  may  show  a  considerable  loss  of  body 
weight,  much  more  than  can  be  accounted  for  by  the  few  days 
starvation.  Upon  the  fourth  or  fifth  day  food  may  be  greedily 
eaten.  After  this  stage  is  reached,  the  ultimate  recovery  of  the 
animal  is  assured.  In  general  nearly  all  dogs  receiving  the  smaller 
dosage  make  complete  recovery  in  spite  of  the  fact  that  during 
the  second  and  third  day  after  the  administration  of  hydrazine 
the  liver  presents,  a  typical,  light  colored  appearance  of  "  fatty 
degeneration."  Bile  may  or  may  not  appear  in  the  urine  and  in 
general  the  urine  contains  no  protein. 

Methods.  In  the  investigations  here  recorded,  hydrazine  (Kahlbaum) 
was  always  introduced  subcutaneously  as  the  sulphate  in  a  2.5  per  cent  solu- 
tion which  is  a  practically  saturated  solution  at  room  temperature.  Esti- 
mations of  the  blood  sugar  content  were  made  according  to  the  method 
indicated  in  a  former  paper,1  the  copper  finally  being  determined  gravimet- 
rically.  Glycogen  in  the  liver  was  determined  by  Pfluger's  method.2 
Blood  pressure  was  recorded  by  a  mercury  manometer  connected  with  a 
femoral  artery.  In  blood  pressure  experiments  narcosis  was  induced  by  a 
mixture  of  morphine  and  atropine  (10  mgms.  morphine  sulphate  and  1  mgm. 
atropine  sulphate  per  kilo  of  body  weight).  Ether  was  not  necessary  after 
the  preliminary  operative  procedures.  Injections  were  made  into  the  femoral 
vein. 

THE  ACTION  OF  HYDRAZINE  UPON  THE  BLOOD  SUGAR  CONTENT. 

It  has  been  previously  intimated  that  there  is  a  paucity  of  liter- 
ature relating  to  the  artificial  production  of  hypoglycaemia.  Aside 
from  the  consideration  of  the  well  known  examples  in  connection 
with  phloridzin  and  uranium  salts,  the  experiments  of  Frank  and 
Isaac3  with  phosphorus  have  shown  for  the  first  time  the  com- 
plete disappearance  of  sugar  from  the  blood  of  rabbits.  A  decrease 
in  blood  sugar  content  has  also  been  observed  by  Porges4  after 
extirpation  of  the  adrenals  in  dogs  and  in  cases  of  adrenal  insuffi- 
ciency caused  by  Addison's  disease.  In  all  these  instances  the 
decreased  sugar  content  of  the  blood  is  accompanied  by  a  rapid 
decrease  or  complete  disappearance  of  the  liver  glycogen  in  spite 
of  the  ingestion  of  relatively  large  quantities  of  carbohydrate. 

1  Underhill:  This  Journal,  i,  p.  113,  1905-06. 

2  Pfluger:  Arch.  f.  d.jes.  Physiol,  cxi,  p.  307,  1906. 

3  Frank  and  Isaac:  loc.  cit. 

4  Porges:  Zeitschr.  f.  klin.  Med.,  lxix,  p.  341,  1909;  lxx,  p.  243,  1910. 


Frank  P.  Underhill 


What  becomes  of  the  glycogen  of  the  liver  and  the  sugar  of  the 
blood  is  at  present  largely  a  matter  of  conjecture.  Whether  the 
carbohydrate  is  merely  transformed  into  some  incompletely  metab- 
olized product  and  the  latter  eliminated,  or  whether  more  rapid 
metabolism  is  induced  resulting  in  increased  carbohydrate  combus- 
tion remain  problems  for  future  determination. 

Experiments  with  Dogs.  Observations  have  been  made  to 
ascertain  the  influence  of  hydrazine  upon  the  blood  sugar  content 
of  dogs.  From  the  data  presented  in  Table  1  it  may  be  seen  that 
an  appreciable  hypoglycaemia  follows  the  introduction  of  hydra- 
zine in  doses  of  50  mgms.per  kilo  of  body  weight,  and  that  twice 
this  quantity  does  not  necessarily  exert  a  more  marked  influence. 
In  a  large  number  of  experiments  hardly  a  single  individual  ani- 
mal has  failed  to  exhibit  this  phenomenon  although  variations  in 
the  degree  of  diminution  may  occur.  The  symptoms  of  extreme 
weakness  in  dogs  after  hydrazine  treatment  may  be  directly  cor- 
related with  the  diminished  store  of  carbohydrate  as  indicated 
by  lowered  blood  sugar  percentage. 

TABLE  1. 


The  Influence  of  Hydrazine  upon  Blood  Sugar  Content  and  Liver  Glycogen 

of  the  Dog. 


NUMBER  OF 
ANIMAL 

SUBCUTANEOUS  INJEC- 
TION OF  HYDRAZINE 
SULPHATE 

SUGAR  CONTENT  OF 
BLOOD 

GLYCOGEN  CONTENT 
OF  LIVER  EXPRESSED 
AS    GRAMS    OF  DEX- 
TROSE 

REMARKS 

mgms.  per  kilo 

per  cent 

1 

50 

0.02 

0.10 

Three  days  after  hydra- 

zine injection. 

2 

100 

0.05 

0.22 

One  day  after  hydra- 

zine injection. 

13 

50 

0.03 

Two  days  after  hydra- 

zine injection. 

15 

50 

0.04 

Two  days  after  hydra- 

zine injection. 

A 

0 

0.14 

Fasted  six  days. 

B 

0 

0.15 

Fasted  six  days. 

C 

0 

0.13 

Emaciated  from  lack  of 

food. 

164      Hydrazine  and  Carbohydrate  Metabolism 

That  starvation  per  se  is  not  responsible  for  the  lowered  blood 
sugar  content  may  be  concluded  from  experiments  A,  B,  and  C, 
in  which  dogs  were  allowed  to  fast  greater  number  of  days  than 
those  in  hydrazine  experiments. 

Experiments  with  Rabbits.  When  hydrazine  is  adminis- 
tered to  rabbits  in  doses  of  50  mgms.  per  kilo  the  animals  refuse 
food  for  a  period  of  at  least  two  days.  No  other  noteworthy 
symptoms  are  conspicuous.  Since  starvation  plays  a  role  in  hydra- 
zine experiments  of  this  type  it  is  imperative  that  the  influence 
of  this  factor  shall  be  determined.  Accordingly  determinations 
have  been  made  of  the  sugar  content  of  the  blood  and  the  glyco- 
gen of  the  liver  in  normal  rabbits  fed  and  kept  under  the  usual 
laboratory  conditions  as  a  comparison  for  similar  determinations 
made  upon  animals  in  every  way  comparable  except  that  all  food 
was  withheld  for  a  period  of  two  days.  This  period  was  chosen 
for  the  reason  that  after  hydrazine  introduction  a  like  period  of 
time  was  allowed  to  elapse  previous  to  killing  the  animals. 

The  results  of  these  trials  (Table  2)  indicate  what  is  gener- 
ally accepted,  namely,  that  the  blood  sugar  content  of  normal 
rabbits  is  approximately  0.10  per  cent,  and  that  usually  the 
liver  contains  a  considerable  quantity  of  glycogen  although 
the  quantity  in  individual  rabbits  may  show  a  noteworthy  varia- 
tion. It  is  also  evident  that  a  period  of  two  days  inanition 
is  sufficient  to  practically  eliminate  the  liver's  store  of  glycogen. 
On  the  other  hand,  the  percentage  of  the  blood  sugar  undergoes 
no  appreciable  change.  When  hydrazine  is  administered  in 
the  doses  indicated  it  is  clear  that  in  the  majority  of  cases  the 
drug  is  capable  of  decreasing  the  sugar  content  of  the  blood,  in 
some  instances  to  a  remarkable  degree,  in  others  only  slightly, 
while  a  practically  normal  blood  sugar  content  is  maintained 
by  a  third  group  of  individuals.  From  this  diversity  of  results 
it  is  obvious  that  the  rabbit  can  not  be  relied  upon  to  invari- 
ably exhibit  hypoglycaemia  after  hydrazine  introduction.  Hence 
results  of  experiments  planned  to  supply  data  of  carbohydrate 
metabolism  from  the  standpoint  of  hyperglycaema  may  be  of 
questionable  value. 

The  problem  as  to  whether  hydrazinized  rabbits  are  capable 
of  maintaining  blood  sugar  content  and  glycogen  store  unchanged 
was  subjected  to  experiment  by  subcutaneously  introducing  dex- 


Frank  P.  Underhill 


165 


TABLE  2. 

The  Behavior  of  Blood  Sugar  Content  and  Liver  Glycogen  in  Rabbits  Treated 

with  Hydrazine. 


NUMBER  OF 
ANIMAL 

SUBCUTANEOUS  INJEC- 
TION OF  HYDRAZINE 
SULPHATE 

SUGAR  CONTENT  OF 
BLOOD 

GLYCOGEN  CONTENT 
OF  LIVER  EXPRESSED 
AS   GRAMS  OF  DEX- 
TROSE 

REMARKS 

mgms.  per  kilo 

per  cent 

10 

0 

0.10 

6.53 

Well-fed  rabbit. 

11 

0 

0.09 

11.07 

Well-fed  rabbit. 

8 

0 

0.12 

0.01 

Two  days  fast. 

9 

0 

0.11 

0.02 

Two  days  fast. 

1 

50 

0.003 

0.06 

No  food  after  hydrazine 

injection. 

2 

50 

0.016 

0.00 

No  food  after  hydra- 

zine injection. 

3 

50 

0.009 

0.02 

No  food  after  hydra- 

zine injection. 

12 

50 

0.08 

0.00 

No  food  after  hydra- 

zine injection. 

18 

50 

0.06 

0.00 

No  food  after  hydra- 

zine injection. 

19 

50 

0.09 

0.00 

No  food  after  hydra- 

zine injection. 

20 

100 

Died  within  five  hours. 

16 

100 

Died    within  twenty- 

four  hours. 

15 

•  50 

0.11 

Subcutaneous  injection 

4  grams  dextrose  per 

kilo  twice  on  day  after 

hydrazine  administra- 

tion. 

trose  into  animals  on  the  day  following  hydrazine  injection. 
On  the  next  day  the  animals  were  killed  and  the  blood  sugar 
content  and  glycogen  of  the  liver  were  determined.  The  results 
of  one  such  experiment  (Rabbit  15,  Table  2)  are  given.  It 
is  obvious  from  what  has  been  said  that  the  exact  interpretation 
of  these  results  is  not  easy  since  from  the  data  furnished  one  is 
unable  to  know  whether  hydrazine  exerted  any  appreciable  influ- 
ence upon  this  animal's  carbohydrate  store.    It  may  be  of  inter- 


1 66      Hydrazine  and  Carbohydrate  Metabolism 


est,  however,  to  point  out  that  in  the  two  experiments,  one  of 
which  is  detailed  in  Table  2,  Rabbit  15,  carried  out  with  this  object 
in  view  both  animals  emitted  sharp  cries  and  exhibited  a  notice- 
able hyperpnoea  immediately  following  the  second  injection  of  dex- 
trose. This  respiratory  disturbance  lasted  about  one  minute,  after 
which  the  animals  appeared  normal. 

To  determine  the  influence  of  small  doses  of  hydrazine  continued 
over  a  considerable  period  of  time,  two  well-fed  rabbits  of  2100 
and  2200  grams  body  weight  respectively  were  selected  and  for 
twelve  days  each  received  25  mgms.  of  hydrazine  sulphate  daily 
without  obvious  detrimental  influence  of  any  kind.  The  only 
effect  noted  was  an  apparent  ravenous  appetite.  After  this 
period  the  dosage  was  increased  to  75  mgms.  daily  for  eighteen 
days  without  any  appreciable  influence.  Body  weight  had  changed 
too  little  to  be  of  significance.  The  animals  were  then  killed. 
The  blood  sugar  content  of  the  two  animals  was  0.07  and  0.12 
per  cent  respectively.  The  glycogen  in  the  liver  amounted  to 
1.56  grams  and  5.1  grams  expressed  as  dextrose. 

In  none  of  the  hydrazinized  rabbits  was  there  any  evidence 
of  the  characteristic  light  colored  liver  seen  with  dogs. 

THE  ACTION   OF   SUBCUTANEOUS  INJECTIONS   OF  DEXTROSE  UPON 
HYDRAZINIZED  DOGS. 

Since  hydrazine  invariably  causes  a  diminution  in  the  per- 
centage of  blood  sugar  it  was  assumed  that  some  light  might  be 
thrown  upon  the  fate  of  the  blood  carbohydrate  by  a  determination 
of  the  assimilation  limits  for  dextrose  injected  subcutaneously 
into  hydrazinized  dogs.  If  dextrose  of  the  blood  undergoes  a 
more  rapid  combustion  in  the  body  subsequent  to  hydrazine 
administration,  one  might  conjecture  that  the  organism  would 
be  capable  of  utilizing  larger  quantities  of  dextrose. 

It  has  been  shown  that  normal  dogs  completely  utilize  dextrose 
injected  hypodermically  in  doses  of  5  grams  per  kilo.1  As  hydra- 
zinized animals  refuse  food  it  is  essential  to  determine  whether 
animals  without  food  for  a  period  of  two  days — the  time  selected 

1  Scott:  Journ.  of  Physiol.,  xxviii,  p.  107,  1902;  Underhill  and  Closson: 
This  Journal,  ii,  p.  117,  1906. 


Frank  P.  Underhill 


167 


for  sugar  introduction  subsequent  to  hydrazine  injections — show 
as  high  assimilation  limits  as  the  normal  animal. 

Three  well-fed  animals  were  selected  for  this  purpose.  Dog  21 
was  a  bitch  of  5.1  kilos.  Dog  22  was  a  dog  of  7.6  kilos.  Dog 
24  was  a  hound  of  15.7  kilos.  To  these  animals,  after  a  prelim- 
inary inanition  period  of  two  days,  subcutaneous  injections  of 
dextrose  were  given  in  doses  of  5  grams  per  kilo.  The  sugar  was 
a  30  per  cent  solution.  For  two  days  subsequent  to  the  sugar 
injection,  urine  was  collected  and  tested  for  dextrose.  In  no  case 
was  there  the  least  trace  of  reducing  substance.  No  food  was 
given  on  the  two  days  following  the  administration  of  dextrose. 

After  a  two  days  period  during  which  the  animals  were  well  fed 
on  a  mixed  diet,  hydrazine  was  subcutaneously  injected  in  doses  of 
50  mgms.  per  kilo  body  weight.  On  the  second  day  following  the 
hydrazine  introduction  quantities  of  dextrose  were  injected  sub- 
cutaneously exactly  equal  to  the  amounts  previously  administered. 
The  sugar  injections  were  all  made  in  the  late  afternoon  and  when 
left  for  the  night  the  animals  were  apparently  in  normal  condition. 
The  next  morning  all  animals  were  found  dead.  The  bladder  of 
Dog  21  contained  13  cc.  of  urine;  37  cc.  of  urine  were  obtained 
from  Dog  22,  while  the  bladder  of  Dog  24  held  70  cc.  of  urine. 
In  no  case  was  there  evidence  of  reducing  substances.  Autopsy 
revealed  nothing  abnormal  except  the  exhibition  of  the  typical 
light  colored  liver. 

Three  experiments  in  every  respect  similar  except  that  the 
preliminary  period  of  starvation  and  sugar  injection  was 
omitted  yielded  results  exactly  comparable.  All  animals  died 
within  a  period  of  twelve  hours  after  the  sugar  injection.  The 
dosage  of  hydrazine  alone  was  not  sufficient  to  cause  the  death 
of  the  animals  in  this  length  of  time,  i.e.,  within  three  days.  In 
fact,  it  has  been  our  experience  that  with  this  dose,  50  mgms. 
per  kilo,  practically  all  animals  recover.  It  is  apparent  that  the 
combination  of  the  two  factors,  hydrazine  and  dextrose,  was  nec- 
essary for  the  unexpected  result. 

In  some  of  the  animals  a  noteworthy  anuria  was  observed  which 
may  be  of  significance  in  the  interpretation  of  the  results  obtained. 
Why  sugar  solutions  thus  introduced  should  exert  such  a  prompt 
toxic  influence  upon  dogs  treated  with  hydrazine  remains  to  be 
ascertained.    This  problem  is  being  subjected  to  further  study. 


1 68      Hydrazine  and  Carbohydrate  Metabolism 


THE  INFLUENCE  OF  HYDRAZINE  UPON  BLOOD  PRESSURE. 

During  the  course  of  this  investigation  it  became  desirable  to 
know  the  action  of  hydrazine  upon  the  arterial  blood  pressure. 
That  hydrazine  exerts  some  influence  in  this  respect  might  be 
concluded  from  the  observation  of  Borissow1  that  hydrazine  at 
first  greatly  increases  the  cardiac  beat  but  subsequently  causes  a  very 
significant  slowing  of  the  heart.  Experiments  to  test  this  point 
have  been  made.  A  tracing  (Plate  III)  is  appended  showing 
that  even  in  relatively  strong  solutions  hydrazine  sulphate  when 
introduced  directly  into  the  circulation  fails  to  exhibit  any  im- 
mediate significant  influence  upon  arterial  blood  pressure. 

In  this  experiment  a  10  kilo  dog  was  employed.  15  cc.  of  0.1 
per  cent,  0.5  per  cent,  1.0  per  cent,  and  2.5  per  cent  hydrazine 
sulphate  solution  were  injected  successively.  The  injections 
lasted  about  one  minute.  In  the  tracing  time  is  recorded  in 
half  seconds. 

CONCLUSIONS. 

Lethal  Dose.  Hydrazine  sulphate  subcutaneously  injected  into 
dogs  and  rabbits  in  doses  of  100  mgms.  per  kilo  invariably  cul- 
minates in  death,  while  all  animals  usually  recover  from  admin- 
istration of  50  mgms.  hydrazine  per  kilo. 

Hypoglycaemia.  Doses  of  50  mgms.  per  kilo  hydrazine  sul- 
phate subcutaneously  injected  into  dogs  leads  to  a  distinct  lowering 
of  the  percentage  of  sugar  in  the  blood;  with  rabbits  this  effect  is 
not  constant. 

During  a  short  period  of  inanition,  dextrose  assimilation  after 
hypodermic  administration  is  as  good  as  in  normal  well-fed  dogs. 

Dextrose  in  doses  of  5  grams  per  kilo  promptly  causes  death 
when  subcutaneously  injected  into  dogs  previously  treated  with 
non-fatal  doses  of  hydrazine. 

Hydrazine  introduced  directly  into  the  blood  stream  shows 
no  appreciable  immediate  influence  upon  arterial  blood  pressure. 

1  Borissow:  loc.  ext. 


rHE  JO 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  X. 


PLATE  III. 


Reprinted  from  The  Journal,  of  Biological  Chemistry,  Vol.  X,  No.  3,  1911 


STUDIES  IN  CARBOHYDRATE  METABOLISM. 
II.  THE  PREVENTION  AND  INHIBITION  OF  PANCREATIC  DIABETES. 

By  FRANK  P.  UNDERHILL  and  MORRIS  S.  FINE. 

(From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  August  16,  1911.) 

The  literature1  relative  to  the  production  of  pancreatic  diabetes 
in  dogs  is  so  well-known  among  physiologists  that  a  detailed 
recital  of  the  conditions  attending  the  induction  of  the  above  men- 
tioned pathological  state  is  unnecessary.  Nevertheless,  for  the 
full  appreciation  of  the  import  of  the  present  communication  it 
may  not  be  out  of  place  to  call  attention  briefly  to  a  few  of  the 
most  salient  features  connected  with  the  production  of  pancreatic 
diabetes.  For  temperamental  and  anatomical  reasons  the  dog 
is  best  suited  for  this  type  of  experimentation.  With  rabbits 
the  production  of  pancreatic  diabetes  may  not  be  constantly 
successful,  owing  to  the  diffuse  distribution  of  the  pancreas  through 
the  mesentery  of  this  animal. 

Accepting  then  as  typical  the  results  obtained  with  the  dog,  one 
may  reasonably  assume  certain  facts  as  fundamental  for  the  pur- 
pose of  further  investigation.  Thus,  it  is  a  matter  of  universal 
experience  that  glycosuria  is  always  provoked  by  the  practically 
complete  removal  of  the  pancreas  in  dogs  whether  the  animals  are 
maintained  in  a  well-fed  or  fasting  condition.  The  time  of  the 
first  appearance  of  sugar  in  the  urine  subsequent  to  the  opera- 
tion in  question  is  not  usually  specifically  stated  in  the  literature 
incident  to  such  experimentation,  hence  such  data  have  been  sup- 
plied in  the  present  paper  under  conditions  strictly  comparable 
to  those  found  to  be  essential  for  the  purposes  aimed  at.  Another 
feature  which  may  be  assumed  to  be  a  constant  factor  in  pancre- 

1cf.  Biedl:  Innere  Secretionen,  Berlin,  1910. 

271 


272         Hydrazine  and  Pancreatic  Diabetes 

atic  diabetes  is  the  accompanying  hyperglycaemia.  Finally, 
the  assumption  may  be  made,  without  meriting  such  universal 
acceptance,  that  pancreatic  diabetes  is  either  identical  with  or 
closely  akin  to  the  pathological  conditions  existing  in  the  diabetes 
of  man. 

Leaving  out  of  consideration  the  decision  as  to  the  identity  of 
experimental  pancreatic  diabetes  with  the  human  abnormal  state 
one  may  reasonably  assume  an  exceedingly  close  relationship 
between  the  two  conditions.  Any  procedures,  therefore,  which 
will  aid  in  the  better  understanding  of  the  experimental  diabetes, 
or  in  the  prevention,  inhibition  or  alleviation  of  the  symptoms,  are 
likely  to  have  an  important  bearing  upon  our  conception  and  treat- 
ment of  the  human  type  of  pathological  carbohydrate  metabolism. 

In  a  previous  paper1  it  has  been  pointed  out  that  the  diamine, 
hydrazine,  is  capable  of  inducing  an  appreciable  hypoglycaemia 
in  dogs.  The  relation  of  the  action  of  hydrazine  to  pancreatic 
diabetes  forms  the  basis  of  the  present  communication. 

EXPERIMENTAL. 

Methods.  Unless  specifically  stated  otherwise  all  operations 
were  carried  out  under  ether  anaesthesia  only.  Aseptic  precau- 
tions were  taken  throughout  the  operative  technique.  After 
operation  the  animals  were  carefully  cared  for,  attention  being 
paid  to  assure  proper  temperature  of  the  animal  room.  After 
death  of  the  animals  operated  upon  for  removal  of  the  pancreas, 
autopsy  revealed  the  practically  entire  absence  of  pancreas  in 
every  instance.  Analysis  of  the  blood  sugar  was  made  according 
to  the  method  usually  employed  in  this  laboratory.2  Sugar  in 
the  urine  was  estimated  with  a  Schmidt  and  Haensch  triple  shadow 
saccharimeter. 

Experiments  to  determine  the  first  appearance  of  sugar  in  the  urine 
of  dogs  subsequent  to  removal  of  the  pancreas. 

In  our  previous  paper  intimation  has  been  made  that  hydrazine 
is  fatally  toxic  in  large  doses  but  that  relatively  small  quantities 

iUnderhill:  This  Journal,  x,  p.  159,  1911. 
2Underhill:  loc.  ext. 


Frank  P.  Underhill  and  Morris  S.  Fine  273 


of  the  drug  exert  only  a  temporary  detrimental  influence.  When 
the  dog  is  subjected  to  a  combination  of  hydrazine  poisoning  and 
pancreas  removal  death  follows  within  a  comparatively  few  hours. 
Therefore  in  order  to  determine  correctly  the  action  of  hydrazine 
upon  the  course  of  events  following  pancreas  removal  it  is  impera- 
tive to  know  the  behavior  of  the  criterion  adopted,  i.e.,  presence 
of  sugar  in  the  urine,  in  animals  deprived  of  the  pancreas  but  with- 
out the  administration  of  hydrazine. 

Two  such  observations  have  been  made  upon  animals  kept 
under  conditions  in  every  way  identical  with  those  depancreatized 
dogs  subjected  to  the  influence  of  hydrazine  to  be  reported  below. 

Experiment  1.  From  a  full-grown  bitch  of  5  kilos  the  pancreas  was 
removed.  The  operation  was  completed  at  4:15  o'clock  on  the  afternoon 
of  May  2.  Upon  catheterization  at  10:00  p.  m.  of  the  same  day  10  cc.  of 
urine  were  obtained  which  contained  approximately  1  gram  of  dextrose. 
The  150  cc.  of  urine  obtained  by  catheterization  the  following  morning  at 
10:30  yielded  5.4  grams  dextrose. 

Experiment  2.  A  bull  bitch  was  depancreatized.  At  12:30  p.  m., 
May  8,  the  operation  had  been  completed.  In  the  100  cc.  of  urine  obtained 
by  catheterization  two  hours  subsequent  to  the  operation  7.33  grams  of 
dextrose  were  eliminated. 

These  observations  indicate  that  dextrose  may  appear  in  signi- 
ficant quantities  in  the  urine  within  a  very  few  hours  after  removal 
of  the  pancreas,  in  some  instances  after  an  interval  of  two  hours. 
It  would  seem  therefore  that  extirpation  of  the  pancreas  has  an 
almost  immediate  effect  in  evoking  glycosuria,  that  there  is  little 
or  no  significant  latent  period,  which  would  conform  to  the  blood 
sugar  content  values  obtained  under  practically  comparable  con- 
ditions.1 These  experiments  also  indicate  that  the  pancreas 
removal  was  sufficiently  complete  to  insure  the  speedy  initiation 
of  pancreatic  diabetes. 

The  prevention  of  pancreatic  diabetes  by  the  subcutaneous  injection 

of  hydrazine. 

The  observation  that  hydrazine  will  almost  invariably  establish 
a  condition  of  hypoglycaemia  in  the  dog  at  once  suggested  the 
possibility  that  by  this  method  pancreatic  diabetes  could  be  pre- 

1  Underhill:  This  Journal,  i,  p.  113,  1905-06. 


274         Hydrazine  and  Pancreatic  Diabetes 


vented.  It  is  well  known  that  during  this  disturbance  of  carbo- 
hydrate metabolism  the  sugar  content  of  the  blood  is  above  the 
normal.  The  assumption  has  been  made  that  in  this  form  of  dia- 
betes sugar  appears  in  the  urine  only  after  the  dextrose  content 
of  the  blood  has  risen  to  a  certain  unknown  point  beyond  which  the 
kidneys  become  permeable  to  the  carbohydrate.  Theoretically 
at  least  if  the  sugar  content  of  the  blood  could  be  kept  below  this 
point  no  dextrose  should  appear  in  the  urine.  In  other  words 
the  appearance  of  sugar  in  the  urine  would  depend  upon  which  of 
the  two  factors  at  issue,  namely,  hydrazine  with  its  hypoglycaemia 
producing  action  or  the  unknown  force  set  free  by  removal  of  the 
pancreas  exerting  an  influence  toward  blood  sugar  accumulation, 
would  display  the  greater  activity. 

With  the  combination  of  pancreas  removal  and  hydrazine  injec- 
tion it  is  therefore  possible  to  imagine  at  least  three  conditions  in 
which  theoretically  glycosuria  may  not  appear.  In  the  first  place, 
the  hydrazine  effect  may  be  very  much  stronger  than  the  influence 
of  pancreas  removal,  in  which  event  sugar  would  not  be  elimin- 
ated in  the  urine.  Secondly,  the  two  effects  may  exactly  neutral- 
ize each  other  causing  a  normal  blood  sugar  content  and  thus  pre- 
vent glycosuria.  Finally,  the  effect  induced  by  removal  of  the 
pancreas  may  be  greater  than  the  hydrazine  influence,  but  the 
action  of  the  latter  may  be  sufficient  to  prevent  accumulation  of 
sugar  in  the  blood  to  the  point  at  which  sugar  elimination  begins. 

These  theoretical  considerations  have  been  put  to  the  test.  The 
details  of  these  experiments  may  be  found  in  the  following  proto- 
cols. 

Experiment  3.  Dog  9.  On  May  9  at  4:00  p.  m.  a  full-grown  terrier 
bitch  of  8  kilos  received  a  subcutaneous  injection  of  16  cc.  of  a  2.5  per  cent 
hydrazine  sulphate  solution  (50  mgms.  per  kilo).  At  11:15  a.  m.,  May  11, 
the  pancreas  had  been  entirely  removed;  the  operation  being  performed 
under  ether  anaesthesia.  The  animal  was  wrapped  in  absorbent  cotton  and 
placed  in  a  cage  in  a  warm  room.  The  urine  (60  cc.)  obtained  by  catheter- 
ization at  3:30  yielded  no  trace  of  reducing  substance  with  Benedict's  solu- 
tion.1 A  similar  negative  result  was  given  with  40  cc.  urine  obtained  by 
catheterization  at  5:15  p.  m.  Upon  catheterization  at  11:30  p.  m.  65  cc. 
of  urine  were  drawn  which  contained  no  dextrose.  When  left  the  dog  was 
in  good  condition  and  responded  to  petting.    The  following  day  at  8:00 


1  Benedict:  This  Journal,  v,  p.  485,  1908. 


Frank  P.  Underhill  and  Morris  S.  Fine  275 


a.  m.  the  animal  was  found  dead.  40  cc.  of  urine  taken  from  the  bladder 
gave  negative  tests  with  Benedict's  solution.  Autopsy  revealed  entire 
absence  of  pancreas;  the  liver  presented  the  typical  pale  appearance  of 
hydrazinized  dogs. 

Experiment  4.  Dog  10.  At  1 :00  p.  m.,  May  13,  20  cc.  of  a  2.5  per  cent 
solution  of  hydrazine  sulphate  were  administered  hypodermically  to  a  well 
nourished  bitch  of  10  kilos.  On  May  15  the  operation  for  pancreas  removal 
was  completed  at  11:30  a.  m.  Immediately  after  the  operation  the  animal 
was  given  a  subcutaneous  injection  of  150  cc.  0.9  per  cent  sodium  chloride 
solution  to  supply  fluid  to  the  tissues.  The  urine  (50  cc.)  obtained  by  cath- 
eterization at  2:30  p.  m.  did  not  reduce  Benedict's  solution.  At  6:00  p.  m. 
catheterization  yielded  45  cc.  urine  which  contained  no  reducing  substance. 
Benedict's  test  was  also  negative  with  the  110  cc.  of  urine  obtained  by  cath- 
eterization at  11:30  p.  m.  The  following  morning  150  cc.  of  urine  obtained 
at  9:00  a.  m.  by  use  of  the  catheter  contained  3.65  grams  dextrose.  The 
animal  was  immediately  given  10  cc.  hydrazine  hypodermically.  At  12:30 
p.m.  the  dog  voided  urine  which  gave  a  faint  test  for  sugar.  The  quantity 
present  was  too  small,  however,  to  be  estimated  with  the  polariscope.  Only 
5  cc.  of  urine  were  obtained  by  catheter  at  3:00  p.  m.  and  this  gave  a  nega- 
tive Benedict's  test.  At  6:00  p.  m.  the  few  cubic  centimeters  of  urine 
obtained  did  not  reduce  Benedict's  solution.  The  animal  at  this  time 
appeared  to  be  failing  rapidly.  It  was  apparent  that  the  kidneys  were 
not  functionating  as  well  as  normally,  therefore,  the  dog  was  given  a  subcuta- 
neous injection  of  100  cc.  0.9  per  cent  sodium  chloride  solution.  The  10  cc. 
of  urine  procured  at  11:15  p.  m.  failed  to  give  any  evidence  of  reducing  com- 
pounds. The  following  morning  the  dog  was  found  dead.  The  bladder 
was  empty.  The  liver  was  very  light  colored.  No  evidence  of  pancreatic 
rests  could  be  seen. 

Experiment  5.  Dog  6.  A  well-fed  bitch  of  11.5  kilos  received  a  sub- 
cutaneous injection  of  23  cc.  of  a  2.5  per  cent  hydrazine  sulphate  solution 
on  the  afternoon  of  April  24.  The  pancreas  was  removed  April  28,  the 
operation  being  completed  at  4:30  p.  m.  A  few  hours  previous  to  the  oper- 
ation 10  cc.  hydrazine  (2.5  per  cent  solution)  were  given.  The  urine  ob- 
tained at  12:30  a.  m.  April  29  did  not  reduce  Benedict's  solution.  At  9:00 
a.  m.  of  the  same  day  the  dog  was  found  dead.  No  reducing  substance 
was  present  in  the  100  cc.  of  urine  found  in  the  cage  bottle.  The  liver 
presented  the  typical  light  colored  appearance.  No  trace  of  pancreas 
was  in  evidence. 

The  results  of  these  observations  warrant  the  conclusion  that 
in  the  quantities  employed  the  subcutaneous  administration  of  hydra- 
zine is  capable  of  preventing  the  appearance  of  sugar  in  the  urine  of 
depancreatized  dogs.  The  non-appearance  of  sugar  in  the  urine 
under  the  experimental  conditions  can  not  be  ascribed  to  a  latent 
period  attendant  upon  pancreas  removal  for  in  the  control  experi- 
ments given  above  glycosuria  promptly  follows  extirpation  of 
the  pancreas. 


276         Hydrazine  and  Pancreatic  Diabetes 


The  sugar  content  of  the  blood  of  hydrazinized  dogs  after  pancreas 

extirpation. 

For  the  correct  interpretation  of  the  above  mentioned  results 
obtained  with  hydrazinized  dogs  after  pancreas  removal  some 
knowledge  of  the  behavior  of  the  blood  sugar  is  imperative.  Such 
data  would  afford  a  much  better  idea  of  the  changes  which  occur 
in  sugar  metabolism  than  can  be  gained  by  a  study  of  the  appear- 
ance of  sugar  in  the  urine  alone.  It  is  true  that  preliminary  exper- 
iments previously  reported  have  demonstrated  the  action  of  hydra- 
zine in  causing  hypoglycaemia  but  it  does  not  necessarily  follow 
that  the  same  activity  prevails  after  pancreas  removal.  It  can 
be  conceived  for  instance  that  hydrazine  may  have  at  least  a  two- 
fold activity — one  causing  a  temporary  hypoglycaemia,  the  other 
an  action  rendering  the  kidney  less  permeable  to  sugar  poured 
into  the  blood  after  pancreas  removal,  in  a  manner  opposite  to 
that  which  has  been  ascribed  to  phloridzin.  Improbable  though 
this  seems,  it  was  necessary  to  test  the  matter  experimentally. 


table  1. 


The  sugar  content  of  blood  of  hydrazinized  dogs  after  pancreas  removal. 


NUMBER  OP  DOG  AND 
BODY  WEIGHT 

SUBCUTANEOUS 
INJECTION  OP 
HYDRAZINE 
SULPHATE 

SUGAR  CONTENT 
OF    BLOOD  IM- 
MEDIATELY BE- 
FORE PANCREAS 
REMOVAL 

SUGAR  CONTENT  OF  BLOOD  AFTER 
PANCREAS  REMOVAL 

Arter 
half  hour 

After 
three  hours 

After 
six  hours 

13.*    12  kilos  

mgms. 
per  kilo 

50 
50 
50 

per  cent 

0.029 

0.09 

0.04 

per  cent 

0.031 
0.15 

per  cent 

0.016 

0.13 

0.03 

per  cent 

0.12 
0.01 

14.*    16.4  kilos  

15.*   8.5  kilos  

'No  sugar  appeared  in  the  urine  throughout  the  experiment. 


In  the  experiments  reported  in  Table  1  the  animals  were  subjected 
to  the  action  of  hydrazine  for  a  period  of  two  days  after  which 
the  pancreas  was  removed.  The  blood  sugar  was  determined  in 
arterial  blood  drawn  from  a  femoral  artery.  The  observations 
reveal  at  least  two  indications:  (a)  Hydrazine  maintains  its  typi- 


Frank  P.  Underhill  and  Morris  S.  Fine  277 


cal  effect  upon  the  blood  sugar  by  keeping  it  far  below  normal  in 
spite  of  the  opposite  accumulative  tendency  characteristic  of 
pancreas  extirpation,  and  (6)  in  animals  in  which  hydrazine  does 
not  appear  to  greatly  reduce  the  blood  sugar,  its  influence  is  still 
sufficient  to  keep  the  content  of  blood  sugar  below  the  point  at 
which  the  kidney  becomes  permeable  to  it. 

In  presenting  these  data  the  realization  is  borne  in  upon  us  that 
the  duration  of  these  experiments  is  not  very  great.  They  repre- 
sent, however,  the  extreme  limit  of  time  that  our  animals  could 
withstand  the  combined  action  of  hydrazine  poisoning,  pancreas 
removal  and  extensive  hemorrhage.  The  latter  alone  is  quite 
detrimental  since  for  exact  determination  of  small  quantities  of 
sugar  in  the  blood  fairly  large  amounts  of  blood  should  be  employed. 
We  have  used  between  twenty  and  thirty  grams  of  blood  for  each 
estimation. 

The  inhibition  of  pancreatic  diabetes  in  dogs  by  subcutaneous  injec- 
tions of  hydrazine. 

The  non-appearance  of  sugar  in  the  urine  of  depancreatized  dogs 
previously  subjected  to  the  influence  of  hydrazine  is  possibly  open 
to  the  criticism  that  the  pancreas  extirpation  was  not  sufficiently 
complete  to  have  invariably  induced  glycosuria.  Although  we 
consider  this  criticism  hardly  valid  inasmuch  as  we  have  never 
failed  to  produce  glycosuria  after  pancreas  removal  in  normal 
dogs  it  is  nevertheless  true  that  the  inhibition  of  pancreatic  dia- 
betes by  hydrazine  would  be  of  far  more  significance  than  the 
prevention  only. 

Details  of  the  experiments  planned  to  demonstrate  this  feature 
of  the  investigation  are  to  be  found  in  the  following  protocols. 

Experiment  6.  Dog  7.  The  pancreas  was  removed  from  a  full-grown 
bitch  of  5  kilos  on  May  2.  The  operation  was  completed  at  4:15  p.  m. 
Urine  collected  by  catheter  at  10:00  p.  m.  contained  about  1  gram  of  dex- 
trose. The  urine  contained  in  cage  bottle  and  that  obtained  up  to  10:30 
a.  m.,  May  3,  was  found  to  contain  5.4  grams  dextrose.  At  this  time  5  cc. 
2.5  per  cent  solution  hydrazine  sulphate  were  injected  subcutaneously.  At 
3:30  p.  m.  50  cc.  of  urine  obtained  by  catheterization  showed  a  content  of 
2.83  grams  dextrose.  A  second  injection  of  hydrazine  (10  cc.  of  the  above 
solution)  was  administered.  The  urine  (250  cc.)  yielded  from  this  time  up 
to  10:30  a.  m.,  May  4,  showed  the  presence  of  1  gram  dextrose.  Cath- 
eterization at  3:30  p.  m.  furnished  45  cc.  of  urine  which  gave  a  very  faint 


278 


Hydrazine  and  Pancreatic  Diabetes 


test  with  Benedict's  solution.  The  quantity  of  dextrose  was  too  small  to 
be  indicated  by  the  polariscope.  The  animal  was  catheterized  at  6:00  p.  m. 
and  at  9:00  p.  m.  and  the  quantity  of  urine  obtained  each  time  consisted 
of  only  a  few  drops  which  gave  a  negative  test  with  Benedict's  solution. 
The  animal  was  found  dead  the  next  morning. 

From  this  experiment  it  is  evident  that  hydrazine  had  a  very 
decided  influence  upon  the  pancreatic  diabetes,  at  first  by  greatly 
diminishing  the  quantity  of  dextrose  eliminated  and  finally  by 
completely  inhibiting  its  appearance  in  the  urine.  This  result 
may  perhaps  be  seen  better  from  the  following  consideration.  The 
sugar  eliminated  from  the  time  of  operation  on  May  2  to  3:30 
p.  m.  on  May  3  amounted  to  9.23  grams,  that  excreted  from  May  3 
at  3:30  p.  m.  to  May  4  at  3:30  p.  m.  was  only  1  gram.  After 
3 :30  p.  m.  on  May  4  the  urine  was  sugar-free.  The  decrease  from 
9.34  grams  of  dextrose  in  approximately  twenty-four  hours  to  1 
gram  during  the  succeeding  day  shows  the  remarkable  rapidity 
with  which  hydrazine  must  accomplish  its  action.  From  the  rapid 
falling  off  in  urine  secretion  it  is  also  evident  that  hydrazine  must 
exert  a  distinct  influence  upon  the  kidney  secretion. 

Experiment  7.  Dog  11.  On  May  16  the  pancreas  was  removed  from 
a  bitch  of  7  kilos  the  operation  being  completed  at  11 :00  a.  m.  As  soon  as 
the  animal  was  under  the  influence  of  ether  preparatory  to  the  operation 
a  subcutaneous  injection  of  50  mgms.  hydrazine  sulphate  per  kilo  was  given. 
Catheterization  at  12:30  p.  m.  yielded  40  cc.  of  urine  which  was  sugar-free. 
The  30  cc.  of  urine  obtained  at  3:30  p.  m.  yielded  a  small  quantity  of  dex- 
trose. At  6:00  p.  m.  the  dog  was  again  catheterized  and  the  20  cc.  of  urine 
yielded  no  reducing  body.  At  this  time  100  cc.  0.9  per  cent  sodium  chloride 
were  injected  subcutaneously.  From  the  catheterization  at  11:20  p.  m. 
30  cc.  of  urine  obtained  gave  no  evidence  of  the  presence  of  dextrose.  On 
the  following  day  100  cc.  of  urine  were  obtained  at  9:00  a.  m.  which  showed 
the  presence  of  the  merest  trace  of  dextrose.  At  this  time  10  cc.  of  hydra- 
zine sulphate  (2.5  per  cent  solution)  were  given.  The  urine  furnished  at 
3 :30  p.  m.  and  5 :30  p.  m.  gave  evidence  of  a  mere  trace  of  reducing  substance. 
At  11:45  p.  m.  the  urine  (10  cc.)  obtained  by  catheterization  furnished  evi- 
dence of  the  presence  of  dextrose  in  the  slightest  degree  only;  by  Benedict's 
test  the  merest  precipitate  formed  on  cooling.  The  animal  was  still  living 
at  10:00  a.  m.,  May  18,  but  was  in  a  somewhat  comatose  condition.  About 
5  cc.  of  urine  obtained  from  the  bladder  contained  no  reducing  substance. 
The  animal  died  during  the  morning. 

The  results  of  this  experiment  demonstrate  that  the  influence 
of  hydrazine  was  almost  sufficient  to  completely  inhibit  the  elim- 


Frank  P.  Underhill  and  Morris  S.  Fine  279 


ination  of  sugar  in  the  urine.  At  one  time  the  inhibition  was 
complete,  but  during  the  night  of  May  16  it  is  evident  that  the 
action  of  hydrazine  was  insufficient  to  overcome  the  sugar  accumu- 
lative power  of  the  depancreatized  dog  and  sugar  was  found  in 
the  urine,  its  appearance  finally  being  prevented  by  a  second  dose 
of  hydrazine. 

Experiment  8.  Dog  25.  From  a  bitch  of  12  kilos  the  pancreas  was 
removed  resulting  in  the  elimination  of  11.20  grams  of  dextrose  for  the  suc- 
ceeding twenty-four  hours.  Then  a  subcutaneous  injection  of  20  cc.  hydra- 
zine sulphate  in  a  2.5  per  cent  solution  was  administered.  Two  hours  later 
the  urine  excreted  held  0.69  gram  dextrose.  An  hour  later  11  cc.  of  urine 
obtained  by  catheterization  yielded  the  merest  trace  of  dextrose,  too  small 
to  be  indicated  by  the  polariscope.  Six  hours  after  the  hydrazine  adminis- 
tration the  urine  was  sugar-free. 

The  data  submitted  justify  the  conclusion  that  hydrazine  admin- 
istered to  dogs  during  pancreatic  diabetes  is  capable  of  completely 
inhibiting  the  elimination  of  sugar  by  the  kidney. 

How  much  hydrazine  is  necessary  to  prevent  pancreatic  diabetes  and 
how  long  does  its  action  persist? 

The  importance  of  the  determination  of  the  quantity  of  hydra- 
zine necessary  to  prevent  pancreatic  diabetes  is  obvious.  How  long 
the  peculiar  action  of  the  drug  endures  is  a  question  also  partic- 
ularly worthy  of  investigation.  These  topics  have  as  yet  been 
considered  in  the  crudest  manner  only  and  hence  the  results  thus 
far  obtained  do  not  allow  us  to  make  more  than  the  most  general 
statements. 

To  find  out  the  limit  of  time  during  which  a  single  subcutaneous 
injection  of  50  mgms.  hydrazine  sulphate  per  kilo  is  still  capable 
of  exerting  its  preventive  action  toward  pancreatic  diabetes,  the 
following  experiment  was  carried  through. 

Experiment  9.  On  June  24  a  12  kilo  bitch  received  the  usual  injection 
of  hydrazine.  Four  days  later  the  pancreas  was  removed.  Within  a  few 
hours  after  the  operation  the  appearance  of  sugar  in  the  urine  in  fairly 
large  quantities  (10  to  12  grams  per  day)  was  noted.  The  animal  lived 
two  days  after  the  operation. 


From  this  result  it  is  apparent  that  a  single  subcutaneous  injec- 
tion of  50  mgms.  hydrazine  sulphate  per  kilo  body  weight  is  not 


28o 


Hydrazine  and  Pancreatic  Diabetes 


capable  of  preventing  the  appearance  of  sugar  in  the  urine  of  dogs 
deprived  of  the  pancreas  four  days  after  the  hydrazine  adminis- 
tration. The  time  during  which  this  influence  prevails  must  lie, 
therefore,  somewhere  between  two  and  four  days. 

A  single  experiment  to  test  the  effect  of  smaller  doses  of  hydra- 
zine is  detailed  in 

Experiment  10.  Dog  26.  On  July  8  a  bitch  of  17.4  kilos  received  a 
subcutaneous  injection  of  25  mgms.  hydrazine  sulphate  per  kilo.  Two  days 
later  the  pancreas  was  removed.  In  this  experiment  it  was  necessary  to 
employ  a  small  quantity  of  morphine  in  order  to  anaesthetize  the  animal. 
The  pancreas  had  been  removed  at  11:30  a.  m.  and  4.3  grams  dextrose  were 
found  in  the  urine  at  2 :30  p.  m.  At  5 :30  p.  m.  the  urine  held  3.2  grams  dex- 
trose. A  second  injection  of  the  same  dose  of  hydrazine  was  now  given. 
The  dog  was  found  dead  at  9:00  a.  m.  the  following  morning.  From  the 
bladder  30  cc.  of  urine  were  obtained  which  reduced  Benedict's  solution. 

This  observation  indicated  that  a  single  injection  of  25  mgms. 
hydrazine  sulphate  per  kilo  is  insufficient  to  prevent  the  elimina- 
tion of  dextrose  in  the  urine  of  dogs  deprived  of  the  pancreas  two 
days  after  the  initial  dose  of  hydrazine. 

A  tentative  hypothesis  to  account  for  the  action  of  hydrazine  upon 

sugar  metabolism. 

Porges1  has  demonstrated  that  adrenal  extirpation  as  well  as 
adrenal  insufficiency  exemplified  in  Addison's  disease  leads  to  a 
significant  hypoglycaemia  and  a  disappearance  of  glycogen  from 
the  liver.  Phosphorus2  produces  the  same  effects  and  Neubauer 
and  Porges3  have  ascribed  to  phosphorus  an  influence  upon  the 
adrenals  leading  to  an  insufficiency  of  adrenal  secretion,  presum- 
ably adrenalin,  thereby  causing  the  liver  glycogen  to  disappear 
and  the  blood  sugar  content  to  decrease.  This  theory  was  based 
upon  experiments  in  which  Neubauer  and  Porges  failed  to  obtain 
evidence  of  adrenalin  in  extracts  of  the  adrenals  after  phosphorus 
administration. 

1  Porges:  Zeitschrift  fur  klinische  Medizin,  lxix,  p.  341,  1910;  lxx,  p.  143. 
2 Frank  and  Isaac:  Archiv  fur  experimentelle  Pathologie  und  Pharmakol- 
ogie,  lxiv,  p.  274,  1911. 

3  Neubauer  and  Porges:  Biochemische  Zeitschrift,  xxxii,  p.  290,  1911. 


Frank  P.  Underhill  and  Morris  S.  Fine  281 

Since  phosphorus  and  hydrazine  are  now  observed  to  be  alike  in 
their  effect  upon  blood  sugar  content  and  liver  glycogen  disappear- 
ance, it  is  reasonable  to  assume  that  the  influence  of  the  two  might 
possibly  be  directed  upon  the  same  object,  namely,  upon  the  adrenal. 
The  explanation  as  to  the  manner  in  which  adrenal  secretion 
influences  carbohydrate  metabolism  is  lacking.  As  a  working 
hypothesis  we  have  made  use  of  the  following  scheme  of  inter- 
action. 

DIAGRAM  INDICATING  INFLUENCE  OF  THE  ORGANS  ON  THE 


METABOLISM  OF  CARBOHYDRATE. 


(Sugar  Content) 
Blood 


Liver 
(Glycogen  Store) 


(Sugar  Content) 
Blood 


(Sugar  Content) 
Blood 


V  Pnncreas  Secretion/ 
s>  < 

/Adrenal  Secretion  \ 


Liver 
(Glycogen  Store) 


PANCREAS  REMOVAL 
(indicated  by  broken  lines) 
leads  to 
Hyperglycaemia 


Anuria 


Diuresis 


No  Glycosuria 


Glycosuria 


282         Hydrazine  and  Pancreatic  Diabetes 

In  the  first  place  we  have  assumed  that  there  is  an  interrelation 
between  the  pancreas,  the  adrenals  and  the  liver  by  the  equilibrium 
of  which  the  sugar  content  of  the  blood  is  kept  practically  constant. 
To  accomplish  this  the  pancreas  is  assumed  to  pour  an  internal 
secretion  into  the  blood  which  tends  to  increase  carbohydrate 
catabolism,  hence  to  diminish  the  blood  sugar.  This  influence 
we  have  called  the  pancreas  secretion  factor.  On  the  other  hand 
the  adrenal  is  assumed  to  give  an  internal  secretion  to  the  blood 
which  has  the  opposite  effect,  namely,  it  has  a  tendency  to  check 
carbohydrate  catabolism,  hence  leads  to  an  accumulation  of 
sugar  in  the  blood.  This  action  we  have  designated  the  adrenal 
secretion  factor.  According  to  this  idea  of  what  occurs  to  keep 
blood  sugar  content  normal,  the  quantity  of  glycogen  which  will 
be  taken  from  the  liver's  store  to  accomplish  this  purpose  is  entirely 
dependent  upon  which  of  the  two  factors  is  predominant  at  any 
given  moment. 

If  we  apply  these  ideas  to  pancreas  extirpation  or  adrenal  re- 
moval, the  effects  which  should  theoretically  follow  according  to 
our  scheme  agree  perfectly  with  recorded  observations  which  for 
the  most  part  have  universal  acceptance. 

When  the  pancreas  has  been  removed  from  an  animal  our  hypoth- 
esis assumes  that  the  pancreas  factor  is  taken  away,  in  other  words 
what  we  have  called  the  influence  that  "  facilitates  carbohydrate 
catabolism"  is  lost.  Consequently  the  blood  sugar  equilibrium 
is  upset  because  the  adrenal  factor,  which  "inhibits  carbohydrate 
catabolism"  is  no  longer  counterbalanced  by  the  pancreas  factor, 
the  influence  normally  furnished  by  the  pancreas.  Without  the 
check  of  the  pancreas  upon  it  the  adrenal  factor  now  has  full  play, 
there  is  less  carbohydrate  catabolism  than  normally,  sugar,  poured 
out  of  the  liver's  store  of  carbohydrate,  accumulates  in  the  blood 
(hyperglycaemia),  diuresis  is  induced  in  the  effort  to  eliminate  the 
sugar  through  the  kidneys,  and  then  glycosuria  is  in  evidence. 
If  this  condition  of  affairs  is  maintained  sufficiently  long  the  liver 
will  be  more  or  less  depleted  of  its  glycogen  owing  to  the  body's 
vain  effort  to  furnish  sufficient  energy  for  its  needs. 

On  the  other  hand,  removal  of  the  adrenals  from  a  normal  ani- 
mal results  in  a  series  of  events  of  the  opposite  order.  The  power 
which  has  a  tendency  to  " inhibit  carbohydrate  catabolism," 
the  adrenal  factor,  has  been  lost,  hence  the  pancreas  factor  holds 


Frank  P.  Underhill  and  Morris  S.  Fine  283 


full  sway,  resulting  in  the  rapid  catabolism  of  all  available  carbo- 
hydrate material  even  to  the  depletion  of  the  liver  glycogen,  leading 
to  hypoglycaemia,  fall  of  blood  pressure,  hence  anuria,  and  no 
glycosuria. 

Extirpation  of  both  pancreas  and  adrenals  should  yield  results 
in  correspondence  with  which  of  the  organs  was  first  removed. 
If  the  adrenals  were  extirpated  first  hypoglycaemia  should  be  in 
evidence;  if  the  pancreas  were  removed  before  the  adrenal,  hyper- 
glycaemia  should  prevail.  The  extirpation  of  both  would  ulti- 
mately mean  a  complete  upset  of  the  sugar  regulation  of  the  organ- 
ism. 

If  we  assume  that  hydrazine  acts  in  a  manner  similar  to  the 
action  ascribed  to  phorphorus  it  is  evident  that  here  hydrazine 
activity  upon  the  blood  sugar  content  would  be  equivalent  to 
partial  or  complete  extirpation  of  the  adrenals.  Hence  according 
to  our  tentative  hypothesis  hydrazine  may  prevent  pancreatic 
diabetes  by  its  inhibition  or  at  least  suppression  of  adrenal  func- 
tion. Viewed  from  another  standpoint  it  is  possible  that  hydra- 
zine has  an  action  upon  sugar  metabolism  entirely  similar  to 
that  exerted  by  the  internal  secretion  of  the  pancreas.  According 
to  this  idea  injections  of  hydrazine  cause  hypoglycaemia  by  in- 
creasing the  efficiency  of  the  pancreatic  secretion  or  by  augment- 
ing its  output.  All  the  results  thus  far  obtained  could  be  inter- 
preted upon  this  basis. 

Assuming  that  hydrazine  does  exert  some  inhibitory  influence 
upon  adrenal  secretion  one  might  expect  perhaps  to  obtain 
some  indication  of  this  by  testing  adrenal  extracts  furnished  by 
hydrazinized  dogs  for  the  generally  accepted  active  principle  of 
adrenal  secretion,  namely,  adrenalin. 

Is  the  secretion  of  adrenalin  inhibited  by  hydrazine  administration? 

The  communication  of  Neubauer  and  Porges1  demonstrating 
the  absence  of  adrenalin  in  the  adrenals  as  a  sequel  to  phosphorus 
administration  would  lead  one  to  infer  that  possibly  hydrazine 
has  a  similar  influence,  more  especially  as  both  these  agents  have 
the  same  action  upon  the  blood  sugar  causing  it  to  be  significantly 
decreased.    The  exhibition2  of  adrenalin  in  the  adrenals  is  usu- 

1  Neubauer  and  Porges:  loc.  cit. 

*cf.  Biedl:  Innere  Secretionen,  for  literature  to  color  reactions. 


284         Hydrazine  and  Pancreatic  Diabetes 


ally  made  by  color  reactions,  among  which  may  be  mentioned  the 
development  of  a  red  color  on  the  addition  of  mercuric  chloride, 
or  green  coloration  caused  by  adding  a  neutral  solution  of  ferric 
chloride  to  aqueous  extracts  of  these  organs. 

In  our  first  attempts  to  gain  evidence  of  the  presence  of  adre- 
nalin in  watery  extracts  of  the  adrenals  by  the  above  mentioned 
color  reactions  we  were  unsuccessful.  It  soon  developed,  how- 
ever, that  the  accompanying  turbidity,  presumably  of  a  protein 
nature,  was  the  cause  for  our  failures,  for  if  the  filtrates  could  be 
obtained  water  clear  the  reactions  proved  to  be  very  delicate.  Our 
final  method  for  demonstrating  the  presence  of  adrenalin  con- 
sisted in  grinding  the  adrenals  to  a  pulp  in  a  mortar  with  fine  sand 
and  a  very  little  water  or  physiological  salt  solution.  This  mix- 
ture was  filtered  and  the  turbid  filtrate  clarified  by  the  addition 
of  just  enough  mercuric  chloride  to  precipitate  the  protein  (usu- 
ally two  or  three  drops  of  a  10  per  cent  solution).  The  clear  fil- 
trate, on  the  addition  of  a  little  more  mercuric  chloride,  soon 
yielded  the  pink  or  red  color  characteristic  of  adrenalin.  The 
development  of  the  coloration  was  much  more  rapid  and  intense 
if  the  solution  was  warmed  somewhat.  The  green  color  reaction 
with  ferric  chloride  was  obtained  in  a  similar  manner,  the  pre- 
liminary addition  of  mercuric  chloride  for  the  purpose  of  clarify- 
ing the  turbid  filtrate  being  without  any  apparent  detriment  to 
the  reaction.  In  fact,  so  little  mercuric  chloride  was  added  that 
it  is  probable  that  practically  all  of  it  was  removed  in  combina- 
tion with  the  precipitate. 

With  this  method  the  extracts  of  the  adrenals  taken  from  seven 
normal  dogs  showed  the  presence  of  significant  quantities  of  adre- 
nalin. 

Dogs  poisoned  with  hydrazine,  the  dose  of  which  was  in  some 
instances  large  enough  to  kill  the  animals  within  twenty-four  hours, 
yielded  adrenals  giving  apparently  just  as  strong  adrenalin  reactions  as 
normal  animals.  This  result  makes  it  evident,  therefore,  that  even  if 
hydrazine  does  have  an  inhibitory  influence  upon  adrenal  produc- 
tion the  inhibition  is  not  complete;  it  is  a  quantitative  influence. 


Frank  P.  Underhill  and  Morris  S.  Fine  285 


CONCLUSIONS. 

Removal  of  the  pancreas  from  normal  dogs  may  be  followed 
by  the  appearance  of  sugar  in  the  urine  within  a  period  of  two  hours. 

Glycosuria  fails  to  manifest  itself  after  pancreas  extirpation  in 
dogs  that  have  received  previous  injections  of  hydrazine.  In 
general  this  effect  is  produced  by  a  single  subcutaneous  injection 
of  50  mgms.  per  kilo  hydrazine  sulphate.  The  influence  of  the 
hydrazine  administration  lasts  between  two  and  four  days.  A 
single  injection  of  25  mgms.  per  kilo  hydrazine  sulphate  does  not 
prevent  sugar  elimination  in  the  urine,  under  the  experimental 
conditions,  after  pancreas  removal. 

The  blood  sugar  content  of  animals  treated  with  hydrazine  and 
then  depancreatized  remains  below  the  normal,  or  at  least  hyper- 
glycaemia  is  not  in  evidence. 

Hydrazine  introduced  into  dogs  during  pancreatic  diabetes  is 
capable  of  completely  inhibiting  the  excretion  of  sugar  by  the 
kidney. 

After  hydrazine  administration  the  presence  of  adrenalin  in 
the  adrenals  may  still  be  demonstrated.  In  this  respect  hydra- 
zine differs  from  phosphorus. 

A  tentative  hypothesis  to  account  for  the  phenomena  is  presented. 

We  are  greatly  indebted  to  Professor  Lafayette  B.  Mendel  for 
aid  in  some  of  the  operations  for  pancreas  extirpation  and  for 
criticism  of  the  manuscript. 


Reprinted  from  the  American  Journal  of  Physiology. 
Vol.  XXVII.  —  January  2,  1911.  — No.  III. 


THE   PRODUCTION   OF   GLYCOSURIA   BY  ADRENALIN 
IN  THYROIDECTOMIZED  DOGS. 

By  FRANK  P.  UNDERHILL, 

[From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.] 

THE  failure  of  adrenalin  to  induce  glycosuria  after  removal  of 
the  thyroids  (the  parathyroids  being  intact)  reported  by  Ep- 
pinger,  Falta,  and  Rudinger  1  was  not  corroborated  by  Underhill  and 
Hilditch.2  In  the  experiments  of  the  latter  adrenalin  called  forth  the 
appearance  of  sugar  in  the  urine  of  all  the  thyroidectomized  animals 
tested. 

In  a  reply  by  Falta  and  Rudinger3  the  following  criticisms  con- 
cerning the  work  of  Underhill  and  Hilditch  are  offered:  (i)  with  a 
single  exception  the  latter  authors  injected  larger  quantities  of  adren- 
alin than  were  employed  by  Eppinger,  Falta,  and  Rudinger.  The 
positive  outcome  of  the  single  exception  is  given  the  doubtful  ex- 
planation that  some  thyroid  secretion  might  still  be  in  circulation 
such  a  short  time  (two  days)  after  thyroidectomy.  On  the  other 
hand,  when  in  the  same  animal  adrenalin  in  the  proper  dosage  called 
forth  glycosuria  eighty-one  days  after  removal  of  the  thyroids,  Falta 
and  Rudinger  point  out  that  "Es  darf  aber  nicht  vergessen  werden, 
dass  bei  Hunden  sich  gar  nicht  so  selten  akzessorische  Schilddriisen 
langs  des  Oesophagus  nach  abwarts  finden,  welche  bis  zum  Pericard 
herabsteigen  konnen." 4  (2)  Falta  and  Rudinger  claim  that  comparison 
can  be  made  with  their  experiments  only  when  the  irritability  of  the 
thyroidectomized  animal  to  an  electrical  current  has  been  determined 

1  Eppinger,  Falta,  and  Rudinger:  Wiener  klinische  Wochenschrift,  1908, 
p.  241;  Zeitschrift  fur  klinische  Medizin,  1908,  lxvi,  p.  1,  and  1909,  lxvii,  p.  1. 

2  Underhill  and  Hilditch:  This  journal,  1909,  xxv,  p.  66. 

3  Falta  and  Rudinger:  Zentralblatt  fur  die  gesamte  Physiologie  und 
Pathologie  des  StorTwechsels,  1910,  xi,  p.  81. 

4  Falta  and  Rudinger:  Loc.  cit.,  p.  83. 

33i 


332 


Frank  P.  Underhill. 


in  an  endeavor  to  discover  latent  tetany.  Underhill  and  Hilditch 
made  no  such  observations.  (3)  Finally,  according  to  Falta  and 
Rudinger,  Underhill  and  Hilditch  made  no  autopsies  to  demonstrate 
whether  perchance  thyroids  or  accessory  thyroids  were  still  present  in 
these  animals. 

In  reply  to  the  criticisms  outlined  above  it  should  be  stated,  first 
of  all,  that  apparently  Falta  and  Rudinger  read  only  a  review5  of 
the  work  by  Underhill  and  Hilditch.  In  the  original  article6  there 
are  given  at  least  two  instances,  out  of  a  possible  four,  in  which  it 
may  be  seen  that  the  same  or  a  smaller  quantity  of  adrenalin  than 
that  employed  by  Eppinger,  Falta,  and  Rudinger  induced  glycosuria 
in  dogs  after  thyroidectomy.  Falta  and  Rudinger  explain  sugar  ex- 
cretion in  the  single  exception  noted  by  them  (Dog  A  7  or  Dog  I)  by 
assuming  that  the  adrenalin  was  administered  too  soon  after  removal 
of  the  thyroids  (two  days).  This  explanation  will  not  suffice,  how- 
ever, for  the  appearance  of  dextrose  in  the  urine  when  adrenalin  was 
injected  according  to  the  Eppinger,  Falta,  and  Rudinger  standard  of 
dosage  in  the  case  of  Dog  D.8  This  injection  occurred  six  days  after 
thyroidectomy,  and  the  statement  is  made  by  the  above-mentioned 
authors  that  at  least  three  days  after  thyroidectomy  adrenalin  failed 
to  produce  glycosuria. 

Falta  and  Rudinger  admit  in  the  case  of  Dog  A  (Dog  I),  in  which 
glycosuria  was  caused  eighty-one  days  after  removal  of  the  thyroids, 
that  there  can  be  no  question  here  of  a  latent  tetany,  but  they  inti- 
mate that  accessory  thyroids  may  be  present.  At  the  time  of  the 
publication  of  our  former  investigation  it  was  considered  undesirable 
to  kill  Dog  A  (Dog  I)  in  order  to  determine  the  presence  of  accessory 
thyroids.  Since  then,  however,  an  autopsy  has  been  performed  upon 
this  animal  with  this  end  in  view.  Every  piece  of  tissue  in  any  way 
bearing  a  resemblance  to  thyroid  tissues  was  carefully  preserved 
and  sent  for  identification  to  Professor  H.  Gideon  Wells  of  the  Uni- 
versity of  Chicago.   In  his  report  concerning  these  tissues  Professor 

5  Underhill:  Zentralblatt  fur  die  gesamte  Physiologie  und  Pathologie  des 
Stoffwechsels,  1909,  x,  p.  641. 

6  Underhill  and  Hilditch:  Loc.  cit. 

7  In  the  article  by  Underhill  and  Hilditch  the  animals  were  called  A,  B,  C, 
etc.  In  the  review  by  Underhill  Dog  A  was  called  Dog  I,  Dog  B  was  designated 
Dog  II,  etc. 

8  Underhill  and  Hilditch:  Loc.  cit. 


The  Production  of  Glycosuria  by  Adrenalin.  333 


Wells  makes  the  statement  that  he  finds  no  evidence  of  thyroid  tissue 
or  accessory  thyroids;  nothing  except  parathyroid  tissue  was  in  evi- 
dence. During  the  present  summer  an  autopsy  was  also  performed 
upon  Dog  C  (Dog  III).  The  result  demonstrated  the  presence  of 
two  somewhat  hypertrophied  parathyroids  but  no  trace  of  thyroid 


TABLE  I. 


Dog. 

Time  after 
thyroidectomy. 

Adrenalin  chloride 
per  kilo  injected. 

Dextrose  in 
urine. 

mgm. 

gm. 

March  3  1.0 

0.0 

A 1 

16  months  j 

March  15  1.0 

4.79 

21 2 

21  days 

1.0 

0.76 

22  3 

4  days 

1.0 

3.67 

23  4 

3  days 

1.0 

14.38 

1  This  animal  was  Dog  A  employed  in  experiments  by  Underhill 
and  Hilditch.   The  dog  never  showed  any  signs  of  abnormality. 
The  body  weight  was  12.1  kilos. 

2  A  dog  of  8  kilos  in  splendid  nutritive  condition.    Both  thyroids 
were  removed,  leaving  two  parathyroids  intact.    Fed  a  mixed  diet. 
Autopsy  revealed  absence  of  thyroid  tissues. 

3  Well-fed  dog  of  13.2  kilos.  No  evidences  of  thyroid  tissues  on 
autopsy. 

4  Dog  of  12  kilos,  in  good  condition.  No  thyroid  tissues  on  autopsy. 
None  of  these  animals  showed  any  abnormality. 

tissue,  nor  could  any  thyroid  tissue  be  found  at  the  autopsy  of  Dog  D. 
The  results  of  the  autopsy  of  Dog  B  (Dog  II)  were  noted  in  our 
former  communication. 

The  criticism  of  Falta  and  Rudinger  concerning  the  points  just 
discussed  are  not  extremely  potent  in  view  of  the  autopsy  findings 
now  reported,  together  with  the  observation  that  all  dogs  gave  glyco- 
suria after  removal  of  the  thyroids  on  treatment  with  adrenalin,  and 
that  two  of  the  four  reacted  positively  with  the  same  dosage  employed 
by  Eppinger,  Falta,  and  Rudinger.  Nevertheless,  in  order  to  decide 
the  question  even  more  definitely  further  experiments  have  been  un- 
dertaken the  results  of  which  may  be  seen  in  Table  I.  The  methods 
employed  were  identical  with  those  outlined  in  our  former  commu- 


334 


Frank  P.  Underhill. 


nication,  except  that  in  the  observations  here  reported  all  adrenalin 
injections  were  made  subcutaneously.  It  may  be  seen  from  these 
data  that  adrenalin  chloride  administered  subcutaneously  to  dogs  in 
the  dosage  of  i  mgm.  per  kilo  body  weight  is  capable  of  provoking 
the  appearance  in  the  urine  of  significant  quantities  of  dextrose, 
three,  four,  and  twenty-one  days,  and  thirteen  months  after  thy- 
roidectomy. In  no  case  were  there  any  abnormal  manifestations, 
nor  could  any  thyroid  tissues  be  found  on  autopsy. 

In  the  present  investigation,  as  in  the  previous  one,  we  have  deemed 
it  unnecessary  to  determine  the  response  of  the  thyroidectomized 
animal  to  electrical  stimulation  in  order  to  discover  latent  tetany. 
The  observation  that  none  of  the  animals  selected  behaved  in  an 
abnormal  manner,  together  with  the  fact  that  Dog  A  (Dog  I)  and 
Dog  C  (Dog  III)  of  our  previous  experiments  lived  more  than  six- 
teen months  without  tetany,  that  like  conditions  obtained  for  Dog  D, 
killed  ten  days  after  thyroidectomy  because  of  an  abscess  at  site  of 
injection,  and  that  Dog  21  of  the  present  investigation  was  allowed 
to  live  more  than  twenty-one  days  after  removal  of  the  thyroids  — 
these  facts  all  speak  against  the  idea  that  latent  tetany  was  present. 

The  criticism  that  adrenalin  injection  was  made  too  soon  after 
operation  in  the  case  of  Dog  A  (Dog  I)  of  our  former  investigation 
will  not  hold  for  our  present  experiments,  since  in  no  case  was  adrenalin 
introduced  under  three  days  after  removal  of  the  thyroids.  A  survey 
of  the  work  of  Eppinger,  Falta,  and  Rudinger  reveals  that  in  several 
instances  their  own  injections  were  made  three  and  four  days  after 
thyroidectomy. 

In  the  paper  by  Falta  and  Rudinger  two  new  experiments 9  are 
detailed  designed  to  corroborate  former  statements.  Concerning  the 
first  experiment  the  question  may  well  be  asked,  "Why  was  the  dose 
of  adrenalin,  10.5  mgm.  (less  than  1  mgm.  per  kilo  for  a  13-kilo  dog) 
divided  into  two  portions  (6  mgm.  and  4.5  mgm.)  and  these  injected 
on  two  separate  days?"  Such  a  procedure  does  not  add  weight 
to  the  statement  of  Eppinger,  Falta,  and  Rudinger  that  1  mgm. 
adrenalin  per  kilo  body  weight  administered  subcutaneously  or  in- 
traperitoneally  into  thyroidectomized  dogs  is  incapable  of  causing 
glycosuria.  The  failure  of  these  quantities  of  adrenalin  to  provoke 
the  appearance  of  sugar  in  the  urine  can  be  duplicated  at  times  in 

9  Falta  and  Rudinger:  Loc  ciL,  p.  82. 


The  Production  of  Glycosuria  by  Adrenalin.  335 

normal  dogs.  In  fact,  even  1  mgm.  per  kilo  often  fails  to  induce 
glycosuria  (see  Dog  22,  Table  II),  and  in  Dog  A  (Dog  I)  without 
thyroids  (see  Table  I)  it  may  be  seen  that  this  dose  failed  on  March 
3,  whereas  on  March  15  it  caused  the  excretion  of  4.79  gm.  dextrose. 
From  our  experience  with  normal  dogs  we  have  been  led  to  the  con- 
clusion that,  although  in  general  1  mgm.  adrenalin  per  kilo  body 
weight  administered  subcutaneously  or  intraperitoneally  is  capable 
of  causing  glycosuria,  animals  are  frequently  encountered  in  which 
this  dose  provokes  no  glycosuria.  On  the  other  hand,  these  same 
animals  at  other  times  behave  in  the  usual  way  and  react  to  doses  of 
1  mgm.  adrenalin  per  kilo. 

The  second  experiment  of  Falta  and  Rudinger  shows  that  5  mgm. 
adrenalin  injected  intraperitoneally  before  removal  of  the  thyroids 
caused  a  slight  glycosuria.  After  thyroidectomy  the  same  quantity 
injected  intraperitoneally  failed.  Later  10  mgm.  introduced  sub- 
cutaneously into  two  places  also  failed  to  provoke  glycosuria.  In 
several  experiments  we  have  noted  the  absence  of  dextrose  in  the 
urine  following  the  intraperitoneal  introduction  of  5  mgm.  adrenalin 
into  normal  dogs  smaller  than  the  one  employed  by  Falta  and 
Rudinger.  The  fact  that  5  mgm.  in  this  instance  failed  to  produce 
glycosuria  does  not  necessarily  mean  that  this  has  been  due  to  thy- 
roid removal.  When  the  subcutaneous  injection  was  given,  the 
animal  weighed  16.2  kilos,  and  yet  only  10  mgm.  adrenalin  were  ad- 
ministered. If  Falta  and  Rudinger  desired  to  offer  evidence  in  sup- 
port of  their  former  statement  that  1  mgm.  adrenalin  per  kilo  will 
not  cause  glycosuria  in  thyroidectomized  dogs,  why  did  they  not 
inject  enough  adrenalin  to  comply  with  their  own  conditions?  Again 
in  a  footnote  the  following  is  given  concerning  the  dog  of  the  second 
experiment:  "Der  Hund  bekam  vor  dem  2.  Versuch  auch  Pituitri- 
num  infundibulare.  Dieses  hat  nach  unseren  Untersuchungen  keinen 
EinfTuss  auf  den  Kohlehydratstoffwechsel."  10  Nevertheless,  in  an 
experiment  the  results  of  which  may  be  so  significant  it  would  have 
been  much  better  to  have  eliminated  this  last  unnecessary  conflict- 
ing factor. 

The  experiments  of  Falta  and  Rudinger  do  not  support  the  original 
statement  of  Eppinger,  Falta,  and  Rudinger  that  1  mgm.  adrenalin 
per  kilo  administered  subcutaneously  or  intraperitoneally  fails  to 

10  Falta  and  Rudinger:  Loc.  cit. 


336 


Frank  P.  Under  hill. 


provoke  glycosuria  in  thyroidectomized  dogs,  since  in  neither  of  the 
protocols  reported  is  there  any  indication  that  these  authors  intro- 
duced i  mgm.  adrenalin  per  kilo  body  weight. 

In  an  article  by  Grey  and  de  Sautelle11  the  conclusion  is  drawn 
that  after  thyroidectomy  in  dogs  glycosuria  evoked  by  adrenalin  is 
much  smaller  than  in  the  normal  animal.  The  method  of  procedure 
adopted  by  these  investigators  was  as  follows :  Dogs  were  kept  upon 
a  fixed  meat  diet  for  several  days.  They  were  then  injected  with 
adrenalin,  after  which  thyroidectomy  was  performed.  During  re- 
covery from  the  operation  a  mixed  diet  was  fed.  Then  meat  was 
again  given  and  adrenalin  administered  a  second  time.  In  the  two 
experiments  recorded  less  sugar  in  the  urine  was  obtained  after  thy- 
roidectomy, as  a  result  of  adrenalin  injection,  than  was  excreted  by 
the  normal  dog. 

The  results  obtained  by  these  authors,  namely,  the  appearance  of 
sugar  in  the  urine  of  thyroidless  dogs  after  administration  of  less 
than  i  mgm.  adrenalin  per  kilo,  stand  in  direct  opposition  to  those 
reported  by  Eppinger,  Falta,  and  Rudinger,  but  they  are  in  perfect 
harmony  with  the  observations  of  Underhill  and  Hilditch.  Grey  and 
de  Sautelle  were  also  unable  to  find  any  thyroid  tissue  at  autopsy. 

On  the  other  hand,  the  experiments  cited  hardly  warrant  the  con- 
clusion drawn  by  the  authors,  namely,  that  after  thyroidectomy  the 
glycosuria,  produced  by  adrenalin  in  the  normal  animal,  is  greatly 
reduced.  The  investigation  was  evidently  carefully  planned  and 
executed,  but  was  based  upon  an  assumption  the  correctness  of  which 
is  questionable.  Consequently  the  conclusion  drawn  is  not  firmly 
established.  From  the  data  presented  it  is  apparent  that  these 
authors  assumed  that  if  the  same  normal  dog  is  kept  under  constant 
conditions  and  given  equal  doses  of  adrenalin  at  two  different  times 
the  quantity  of  sugar  excreted  after  these  injections  should  be  approxi- 
mately the  same.  If  that  assumption  was  not  made,  then  the  ex- 
periments are  purposeless.  All  experimental  evidence,  however, 
points  against  such  an  assumption,  for  the  same  normal  dog  under 
constant  conditions  does  not  necessarily  excrete  the  same  quantity 
of  sugar  with  the  same  dosage  of  adrenalin  given  at  two  different 
times.    If  a  normal  dog  will  not  invariably  respond  to  adrenalin 

11  Grey  and  de  Sautelle:  Journal  of  experimental  medicine,  1909,  xi,  p.  659 # 


The  Production  of  Glycosuria  by  Adrenalin,  337 

twice  alike,  it  is  fallacious  to  attribute  a  lessened  elimination  of  sugar 
to  the  loss  of  the  thyroids  without  at  least  the  support  of  a  great 
number  of  experiments  all  showing  the  same  marked  tendency.  In 
an  experiment  having  as  its  object  the  study  of  the  influence  of  the 
thyroids  upon  carbohydrate  metabolism,  it  is,  therefore,  apparent  that 
quantitative  changes  in  sugar  excretion  should  be  given  little  weight. 

As  corroboratory  to  the  views  just  expressed  the  data  in  Table  II 
are  submitted.  In  these  experiments  the  plan  followed  was  very 
similar  to  that  outlined  by  Grey  and  de  Sautelle,  and  although  the 
details  differ  somewhat  the  end  aimed  at,  to  keep  conditions  of  diet 
constant,  was  attained.  The  animals  had  been  fed  upon  meat  for 
several  days  before  the  experiments  began.  They  were  then  fed 
upon  a  constant  mixed  diet  for  a  period  of  five  days.  Then  adrenalin 
was  given  subcutaneously,  —  1  mgm.  per  kilo  body  weight.  On  the 
day  of  the  injection  the  usual  quantity  of  water  was  given  but  no 
food.  As  a  rule  sugar  elimination  ceased  within  twenty-four  hours 
after  adrenalin  administration.  The  dogs  were  then  fed  upon  meat 
until  five  days  before  the  second  adrenalin  injection.  During  these 
five  days  the  mixed  diet  was  again  given.  As  before,  no  food  was 
offered  on  the  injection  day.  After  the  second  adrenalin  administra- 
tion the  thyroids  were  completely  removed,  but  at  least  two  para- 
thyroids, one  on  each  side,  were  left  intact.  This  was  confirmed  at 
autopsy.  During  the  period  of  recovery  from  the  operation  the  dogs 
received  the  meat  diet.  Five  days  before  the  third  adrenalin  injec- 
tion the  mixed  diet  was  fed,  and  as  previously  no  food  was  given  on 
the  day  of  injection.  By  such  a  regime  the  animals  remained  prac- 
tically constant  in  weight.  In  every  instance  1  mgm.  adrenalin  per 
kilo  was  administered  subcutaneously  in  the  region  of  the  lower  ribs. 
The  subcutaneous  injection  possesses  the  following  advantages: 
animals  do  not  die  so  frequently  as  with  the  intraperitoneal  injection, 
and  are  not  so  likely  to  vomit  or  have  diarrhoea  or  bloody  urine.  In 
this  particular  point  we  differ  from  Grey  and  de  Sautelle,  since  their 
injections  were  made  intraperitoneally.  The  principle,  however, 
is  identical  in  the  two  cases,  since  the  mechanism  involved  in  each 
case  is  the  same.  Furthermore,  there  is  no  basis  for  assuming  that 
adrenalin  given  intraperitoneally  will  show  a  different  behavior 
concerning  the  point  under  discussion  than  adrenalin  administered 
subcutaneously. 


338 


Frank  P.  Underbill. 


The  data  presented  in  Table  II  demonstrate  conclusively  that 
adrenalin  administered  twice  to  the  same  normal  animal  under  like 
conditions  does  not  necessarily  provoke  the  same  degree  of  glycosuria 
in  the  two  instances.  Moreover,  in  every  case  reported  adrenalin 
induced  glycosuria  after  thyroidectomy,  and  the  quantity  of  sugar 


TABLE  II. 
Sugar  in  Urine  in  Grams. 


Dog. 

Before  thyroidectomy. 

After 

First  injection. 

Second  injection. 

thyroidectomy. 

A 
Xi 

1.27 

...  | 

0.16  | 

March  3  0.0 
March  15  4.79 
Died  during 
operation 

21 

9.70 

1.20 

0.76 

22 

0.0 

3.40 

3.67 

23 

1.22 

4.64 

14.38 

1  This  animal  was  a  dog  of  7.0  kilos.  The  details  concerning  the 
other  dogs  are  given  in  the  footnotes  of  Table  I,  p.  333.  In  each  ex- 
periment 1  mgm.  adrenalin  per  kilo  was  injected  subcutaneously. 


eliminated  after  the  operation  was  not  uniformly  decreased.  In 
fact,  in  two  of  three  experiments  detailed  more  sugar  was  excreted  by 
the  thyroidectomized  dog  than  appeared  in  the  urine  of  the  normal 
dog. 

Conclusions. 

Renewed  investigation  concerning  the  efficiency  of  adrenalin  in 
provoking  glycosuria  in  thyroidectomized  dogs  leads  to  a  reiteration 
of  our  former  conclusion  that  adrenalin  chloride  administered  sub- 
cutaneously in  doses  of  i  mgm.  per  kilo  body  weight  causes  a  signifi- 
cant glycosuria  in  dogs  deprived  of  both  thyroids  but  retaining  at 
least  two  parathyroids.  The  criticisms  of  Falta  and  Rudinger  with 
respect  to  our  former  experiments  have  in  no  way  invalidated  this 
conclusion. 


The  Production  of  Glycosuria  by  Adrenalin,  339 

In  the  investigcation  by  Falta  and  Rudinger,  put  forth  in  support 
of  the  conclusions  deduced  by  Eppinger,  Falta,  and  Rudinger,  they 
have  failed  to  comply  with  the  conditions  laid  down  by  the  latter. 
Consequently  the  results  offered  by  Falta  and  Rudinger  cannot  be 
accepted  as  proof  that  adrenalin  administered  to  thyroidectomized 
dogs  in  doses  of  1  mgm.  per  kilo  is  incapable  of  causing  glycosuria. 

The  observations  of  Grey  and  de  Sau telle  are  in  harmony  with  our 
own  results  and  stand  in  direct  opposition  to  the  position  taken  by 
Eppinger,  Falta,  and  Rudinger.  The  validity  of  the  conclusions 
drawn  by  Grey  and  de  Sautelle,  however,  may  be  questioned,  since 
the  investigation  was  based  upon  an  assumption  the  correctness  of 
which  has  not  yet  been  established. 

Experiments  are  reported  demonstrating  that  adrenalin  admin- 
istered subcutaneously  to  normal  dogs  in  doses  of  1  mgm.  per  kilo 
causes  a  widely  varying  degree  of  glycosuria.  The  same  quantity  of 
adrenalin  introduced  into  thyroidectomized  dogs  under  like  condi- 
tions is  capable  of  inducing  as  great  or  even  a  greater  glycosuria  than 
occurs  with  the  normal  animal. 


Reprinted  from  the  Proceedings  of  the  Society  for  Experimental  Biology  and  Medicine, 

1911,  viii,  pp.  126-127. 


76  (60l) 

The  behavior  of  fat-soluble  dyes  in  the  organism. 

By  LAFAYETTE  B.  MENDEL  and  AMY  L.  DANIELS. 

[From  the  Laboratory  of  Physiological  Chemistry,  Sheffield 
Scientific  School,  Yale  University,  New  Haven,  Conn.] 

It  is  well  known  that  the  fat-soluble  dye,  Sudan  III.,  is  readily 
deposited  in  the  adipose  tissue  of  animals.  An  attempt  was  made 
by  the  authors  to  study  the  movements  of  the  dye  under  conditions 
where  fat  transport  takes  place  (e.  g.,  in  starvation,  phlorhizin- 
and  phosphorus  poisoning).  The  dye  readily  migrates  into  the 
blood  with  the  fat  under  these  conditions,  but  is  rarely  found  in  the 
liver  tissue  into  which  large  quantities  of  fat  enter  (fatty  infiltra- 
tion). This  is  explained  by  the  observation  that  the  Sudan  III. 
is  abundantly  excreted  with  the  bile  into  the  intestine  from  which 
it  may  be  reabsorbed.  Sudan  III.,  which  is  insoluble  in  water, 
is  not  excreted  through  the  kidneys  except  where  alimentary 
lipuria  is  induced  (in  rabbits  and  rats).  The  elimination  from 
the  liver  is  not  accomplished  through  the  solvent  medium  of  fat 
excreted  in  the  bile  (lipocholia) ;  but  the  dye  is  soluble  in  bile  as 
well  as  in  solution  of  the  isolated  bile  salts.  We  have  thus  estab- 
lished a  path  of  elimination  for  fat-soluble  (or  bile-soluble)  substances 
through  the  biliary  secretion.  An  investigation  of  a  considerable 
number  of  water-insoluble,  fat-soluble  compounds — mostly  non- 
toxic aniline  dyes  and  food  colors — showed  comparable  conditions 
justifying  the  above  general  conclusion.  It  has  further  been 
established  that  these  water-insoluble  compounds  do  not  experi- 
ence absorption  from  the  intestine  in  the  absence  of  bile.  Dis- 
solved in  fat-emulsion  and  introduced  into  the  organism  by  ali- 
mentary, subcutaneous,  or  intravenous  paths,  these  dyes  are  al- 
ways eliminated  with  the  bile  into  the  intestine.  When  there  is 
a  paucity  of  fat  in  the  diet  the  fat-soluble  dyes  may  be  absorbed 
through  the  agency  of  reabsorbed  bile,  but  they  are  speedily 


Scientific  Proceedings  (44). 


2 


eliminated  again  by  the  liver  channels;  with  an  abundance  of 
fat  to  act  as  carrier,  they  travel  with  it  through  the  lymphatics 
into  the  circulation.  The  distribution  of  fat-soluble  dyes  within 
the  organism  depends  on  the  presence  of  fat  and  its  migrations. 
Thus  they  may  be  carried  to  or  from  adipose  tissues,  be  deposited 
in  the  egg-yolk,  or  be  secreted  in  company  with  fat  in  the  milk 
of  animals;  they  apparently  do  not  traverse  the  placenta.  The 
dyes  have  not  been  detected  in  the  lipoids  of  the  nervous  tissue. 
We  have  failed  to  note  any  inability  on  the  part  of  animals  to 
utilize  fats  in  which  Sudan  III.  has  been  deposited. 


Reprinted  from  the  Proceedings  of  the  Society  for  Experimental  Biology  and  Medicine, 

191 1,  viii,  p.  80. 


47  (572) 

The  production  of  glycosuria  as  a  result  of  the  intravenous 
injection  of  Witte's  peptone. 

By  YANDELL  HENDERSON  and  FRANK  P.  UNDERHILL. 

[From  the  Physiological  Laboratory,  Yale  Medical  School  and  the 
Sheffield  Laboratory  of  Physiological   Chemistry,  Yale 
University.] 

Renewed  investigation  concerning  the  phenomena  provoked 
by  intravenous  administration  of  Witte's  peptone  has  demon- 
strated the  production  of  a  marked  glycosuria,  following  such  in- 
jections. The  appearance  of  sugar  in  the  urine  is  accompanied  by 
hyperglycemia.  The  experiments  were  carried  out  for  the  most 
part  upon  dogs.  When  Witte's  peptone  is  injected  into  the  rabbit 
glycosuria  is  not  in  evidence,  an  observation  which  is  in  entire 
accord  with  the  failure  of  this  substance  to  induce  certain  other 
phenomena  in  this  animal  which  are  brought  about  in  the  dog. 

The  tentative  hypothesis  is  advanced  that  the  presence  of  sugar 
in  the  urine  is  induced  as  a  result  of  the  respiratory  disturbances 
set  up  by  the  "peptone"  injection. 

The  phenomena  connected  with  the  injection  of  "peptone" 
mixtures  are  being  subjected  to  further  investigation. 


Reprinted  from  the  American  Journal  of  Physiology. 
Vol.  XXVII.  —  February  1,  1911. —No.  IV. 


THE  METABOLISM  OF  DOGS  WITH  FUNCTIONALLY 
RESECTED  SMALL  INTESTINE. 


By  FRANK  P.  UNDERHILL. 

(With  the  Collaboration  of  CHESTER  J.  STEDMAN  and  JESSAMINE  CHAPMAN.) 

[From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.] 

r  I  AHE  necessity  of  removing  varying  lengths  of  intestine  from 
man  has  made  imperative  extensive  investigations  concerning 
the  influence  of  such  surgical  procedures  upon  nutritional  processes. 
This  is  particularly  true  with  respect  to  the  mechanisms  involved  in 
digestion  and  absorption.  For  obvious  reasons  the  experimental 
demonstration  of  the  effects  incident  to  resection  of  portions  of  the 
enteric  tract  has  been  made  for  the  most  part  upon  the  dog  and  cat. 
Of  particular  importance  in  this  connection  have  been  the  observa- 
tions recorded  by  Harlay,1  Senn,2  Trzebicky,3  Monari,4  De  Filippi,5 
Erlanger  and  Hewlett,6  Flint,7  and  others.8  From  these  investiga- 
tions it  may  be  stated  that  extirpation  of  portions  of  the  small  intes- 
tine generally  entails  a  decreased  absorption  of  the  nitrogenous  and 
fatty  constituents  of  the  food.  The  extent  of  lessened  absorption 
depends  upon  the  relative  length  of  intestine  removed,  and  also  upon 
the  period  which  has  elapsed  after  the  operation,  that  is,  whether 
compensation  has  been  established.    The  composition  of  the  food 

1  Harlay:  Proceedings  of  the  Royal  Society,  London,  (B),  1899,  lxiv,  p.  255. 

2  Senn:  Experimentelle  Beitrage  zur  Darmchirurgie,  Basle,  1892,  quoted. 

3  Trzebicky:  Archiv  fur  klinische  Chirurgie,  1894,  xlviii,  p.  54. 

4  Monari:  Beitrage  zur  klinischen  Chirurgie,  1896,  xvi,  p.  479. 

5  De  Filippi:  Archives  italiennes  de  biologie,  1894,  xxi,  p.  445. 

6  Erlanger  and  Hewlett:  This  journal,  1901,  vi,  p.  1. 

7  Flint:  Transactions  Connecticut  State  Medical  Society,  1910,  p.  283.  A 
complete  discussion  of  the  earlier  literature  upon  this  subject  both  in  connection 
with  the  human  subject  and  with  the  lower  animals  may  be  found  in  this  article. 

8  London  and  Dmitriew:  Zeitschrift  fur  physiologische  Chemie,  1910,  lxv, 
p.  213;  Carrel,  Meyer,  and  Levene:  This  journal,  1910,  xxv,  p.  439. 

366 


The  Metabolism  of  Dogs. 


3fy 


may  play  a  significant  role,  since  it  has  been  established  that  large 
quantities  of  fat  bring  about  a  much  poorer  utilization  of  food  nitro- 
gen than  occurs  when  smaller  quantities  of  fat  are  ingested.  Dimin- 
ished utilization  of  fat  is  particularly  noticeable  in  these  experimental 
animals.  With  respect  to  carbohydrate  absorption  the  observations 
are  somewhat  at  variance,  since  in  some  instances  it  has  been  reported 
that  the  faeces  held  reducing  substances,  and  in  other  experiments 
none  were  found. 

In  the  investigation  to  be  reported  study  of  three  problems  was 
held  in  view,  (1)  the  absorption  of  foodstuffs  after  functional  removal 
of  varying  lengths  of  small  intestine,  (2)  a  study  of  food  absorption  at 
different  intervals  after  the  operation,  and  (3)  the  determination  of 
carbohydrate  utilization  under  varied  conditions. 

Experimental. 

Description  of  the  animals  employed.  —  The  dogs  used  in  these  ex- 
periments were  placed  at  our  disposal  through  the  kindness  of  Pro- 
fessor Joseph  Marshall  Flint,  and  for  convenience  will  be  designated 
Dog  A,  Dog  B,  and  Dog  No.  12.  In  these  three  instances  a  portion 
of  the  small  intestine  was  short-circuited. 

Dog  A  was  a  water  spaniel  of  8.3  kilos  in  splendid  nutritive  condi- 
tion. Two  weeks  after  the  operation,  when  she  came  into  our  posses- 
sion, January  26,  1910,  the  wound  was  well  healed.  The  stools  of  this 
animal  were  fairly  well  formed,  and  throughout  the  entire  period  of 
observation  diarrhoea  was  not  once  noticed.  On  autopsy  about  nine 
months  later  it  was  found  that  the  entire  intestine  was  412.5  cm.  long 
and  that  162.5  cm.  or  39  per  cent  had  been  short-circuited. 

Dog  B  was  a  mongrel  bitch  weighing  13.5  kilos  when  she  came  into 
our  possession,  January  26,  1910.  The  animal  was  in  fair  nutritive 
condition,  but  suffered  from  persistent  diarrhoea,  discharging  copious 
liquid  stools  of  exceedingly  foul  odor.  On  her  entrance  to  the  labora- 
tory two  weeks  after  the  operation  the  wound  was  well  healed.  On 
autopsy  about  nine  months  later  measurement  showed  the  entire 
length  of  intestine  to  be  525  cm.,  of  which  350  cm.  had  been  short- 
circuited,  —  66  per  cent. 

Dog  No.  12  was  the  animal  called  Dog  No.  12  in  Professor  Flint's 
report.    She  was  received  into  the  laboratory  January  8,  1909,  al- 


368 


Frank  P.  Under  hill. 


most  six  months  after  the  operation.  At  that  time  she  weighed  7.4 
kilos;  a  distinct  loss  of  weight  which  was  never  regained.  Diarrhoea 
was  persistent  and  the  appetite  was  ravenous.  On  autopsy  it  was 
found  that  of  the  324  cm.  of  small  intestine  235  cm.  or  73  per  cent 
had  been  short-circuited. 

Methods.  —  In  general  the  methods  followed  were  those  usually 
employed  in  metabolism  experiments  in  this  laboratory.  Urine  was 
collected  in  twenty-four-hour  periods  by  catheterization.  The  water 
content  of  the  faeces  was  determined  by  drying  them  upon  the  water 
bath  under  acid  alcohol  and  preserving  them  air-dry.  Carbohydrates 
in  the  faeces  were  estimated  according  to  the  method  of  Tsuboi.9 
Food  was  given  in  two  portions  daily.  One  meal  was  at  nine  o'clock 
in  the  morning,  the  other  at  four  o'clock  in  the  afternoon. 

Description  of  Experiment  I. 

In  this  experiment  Dog  A  and  Dog  B  were  employed.  For  several 
days  previous  to  the  investigation  the  animals  had  received  an  ade- 
quate mixed  diet.  The  experiment  was  planned  to  determine  the 
ability  of  these  dogs  to  maintain  nitrogenous  equilibrium  upon  suffi- 
cient mixed  diets  the  composition  of  which  was  to  be  radically  altered 
at  intervals  with  respect  to  the  content  of  fat  and  carbohydrate.  As 
originally  planned,  each  period  of  the  experiment  was  to  extend  over 
five  days.  Owing  to  faecal  contamination  of  the  urine  of  Dog  B,  it 
was  practically  impossible  to  obtain  urine  and  faeces  unmixed  for  five 
consecutive  days,  with  the  exception  of  the  first  period.  Accordingly 
in  the  other  periods  balances  have  been  made  covering  the  consecu- 
tive days  on  which  the  urine  was  uncontaminated.  Although  it  was 
out  of  the  question  at  times  to  include  the  excreta  in  the  balance, 
food  was  given  as  usual  and  all  other  conditions  remained  constant, 
so  that  when  a  balance  of  less  than  five  days  is  reported  the  results 
are  probably  fairly  representative  of  the  animal's  condition  through- 
out the  entire  period.  The  periods  of  this  experiment  have  been 
designated  Periods  1,  2,  3. 

Diets.  Dog  A.  —  The  diet  for  Period  1  consisted  of  100  gm. 
meat,  50  gm.  cracker  meal,  20  gm.  lard,  10  gm.  bone  ash,  and  150  c.c. 

9  Tsuboi:  Zeitschrift  fur  Biologie,  1897,  xxxv,  p.  68. 


The  Metabolism  of  Dogs. 


369 


water,  the  total  nitrogen  content  of  which  amounted  to  4.39  gm., 
or  56  gm.  nitrogen  per  kilo.  The  estimated  fuel  value  was  505 
calories,  or  64  calories  per  kilo. 

In  Period  2  the  diet  was  made  up  of  100  gm.  meat,  75  gm.  cracker 
meal,  36  gm.  lard,  10  gm.  bone  ash,  and  230  c.c.  water,  containing 
4.81  gm.  nitrogen  and  750  calories,  or  0.62  gm.  nitrogen  and  96 
calories  per  kilo. 

The  composition  of  the  food  in  Period  3  was  as  follows:  100  gm. 
meat,  100  gm.  cracker  meal,  10  gm.  bone  ash,  and  300  c.c.  water.  The 
diet  contained  5.23  gm.  nitrogen,  and  a  calculated  fuel  value  of  520 
calories,  or  0.69  gm.  nitrogen  and  67  calories  per  kilo  body  weight. 

Further  details  concerning  Experiment  I,  Dog  A,  may  be  found  in 
Tables  I  and  III,  pages  370  and  372  respectively. 

Diets.  Dog  B.  —  In  the  first  period  this  dog  was  placed  upon  a 
diet  consisting  of  200  gm.  meat,  80  gm.  cracker  meal,  30  gm.  lard, 
20  gm.  bone  ash,  and  300  c.c.  water.  This  diet  contained  8.44  gm. 
nitrogen  and  had  an  estimated  fuel  value  of  837  calories,  or  0.63  gm. 
nitrogen  and  63  calories  per  kilo  body  weight. 

In  Period  2  the  diet  was  made  up  of  200  gm.  meat,  120  gm.  cracker 
meal,  50  gm.  lard,  30  gm.  bone  ash,  and  400  c.c.  water,  and  contained 
9. 1 1  gm.  nitrogen  and  n 80  calories  (calculated),  or  0.71  gm.  nitrogen 
and  93  calories  per  kilo. 

The  diet  in  Period  3  had  the  following  composition:  200  gm.  meat, 
160  gm.  cracker  meal,  30  gm.  bone  ash,  and  450  c.c.  water.  The  food 
contained  9.78  gm.  nitrogen  with  an  estimated  fuel  value  of  875 
calories,  or  0.79  gm.  nitrogen  and  70  calories  per  kilo  body  weight. 
For  further  details  of  Experiment  1,  Dog  B,  see  Tables  II  and  III, 
pages  371  and  372  respectively. 

Discussion  of  Results  of  Experiment  I. 

Throughout  the  entire  first  experiment,  Dog  A  maintained  body 
weight  and  furnished  large  positive  nitrogen  balances  (see  Tables  I 
and  III).  Nitrogen  utilization  was  practically  the  same  as  that  of  a 
normal  animal  upon  a  mixed  diet.  The  effect  of  increasing  carbohy- 
drate and  fat  intake  had  little  influence  upon  nitrogen  equilibrium, 
although  fat  utilization  on  this  diet  was  somewhat  diminished.  A 
further  increase  is  to  be  noted  when  lard  was  entirely  removed  from 


370  Frank  P.  Underbill. 

TABLE  I 

Experiment  I.  —  Dog  A,  39  per  Cent  Intestine  Resected.1 


Period  1.  —  Food:  100  gm.  Meat;  50  gm.  Cracker  Meal;  10  gm.  Lard;  10  gm. 
Bone  Ash;  150  c.c.  Water. 

Nitro- 
gen in 
food. 

Urine. 

Faeces. 

Date. 

Body 
weight. 

Vol- 
ume. 

Total 

Weight. 

Water 

Nitro- 
gen. 

Ether 
extract. 

nitro- 
gen. 

Moist 

Air- 
dry. 

con- 
tent. 

1910. 

Jan.  29 

kilos. 

7.8 

gm. 

4.39 

c.c. 

160 

gm. 

1.95 

gm. 

3.3 

gm. 

2.7 

per  cent. 

18 

gm. 

gm. 

"  30 

7.7 

4.39 

160 

3.06 

"  31 

7.7 

4.39 

180 

3.15 

29.0 

>  2.66 

11.96 

Feb.  1 

7.7 

4.39 

100 

2.70 

36.5 

22.5 

38 

"  2 

7.7 

4.39 

100 

3.21 

59.6 

38.0 

36 

Period  2.  —  Food:  100  gm.  Meat;  75  gm.  Cracker  Meal;  36  gm.  Lard;  10  gm. 
Bone  Ash;  230  c.c.  Water. 

Feb.  3 

7.7 

4.81 

200 

3.21 

"  4 

7.7 

4.81 

200 

2.93 

77.2 

46.7 

39 

"  5 

7.7 

4.81 

200 

4.08 

26.8 

>  3.68 

27.35 

"  6 

7.7 

4.81 

200 

3.03 

40.8 

21.0 

48 

"  7 

7.7 

4.81 

200 

3.43 

53.8 

28.7 

46 

Period  3.  —  Food:  100  gm.  Meat;  100  gm.  Cracker  Meal;  10  gm.  Bone  Ash; 

300  c.c.  Water. 

Feb.  8 

7.7 

5.23 

230 

4.26 

36.8 

17.9 

51 

- 

"  9 

7.6 

5.23 

240 

4.02 

55.6 

26.0 

53 

"  10 

7.6 

5.23 

240 

3.90 

>  3.67 

9.80 

"  11 

7.7 

5.23 

280 

3.84 

32.6 

20.4 

37 

"  12 

7.7 

5.23 

310 

4.26 

97.5 

43.7 

55 

1  In  all  experiments  the  urine  showed  an  acid  reaction  to  litmus. 

The  Metabolism  of  Dogs.  371 


table  n. 

Experiment  I.  —  Dog  B,  66  per  Cent  Intestine  Resected. 


Period  1.  —  Food:  200  gm.  Meat;  80  gm.  Cracker  Meal;  30  gm.  Lard; 

20  GM. 

Bone  Ash;  300  c.c.  Water. 

Urine. 

Faeces. 

Body 
weight. 

Nitro- 
gen in 
food. 

Date. 

Vol- 
ume. 

Total 

Weight. 

Water 

Nitro- 
gen. 

Ether 
extract. 

nitro- 
gen. 

Moist. 

Air- 
dry. 

con- 
tent. 

1910. 

Jan.  29 

kilos 

13.3 

gm. 

8.44 

c.c. 

550 

gm. 

9.24 

gm. 

23.9 

gm. 

9.7 

per  cent. 

59 

gm. 

gm. 

"  30 

13.0 

8.44 

350 

6.90 

93.8 

52.7 

44 

"  31 

13.0 

8.44 

420 

5.40 

104.7 

50.2 

52 

>  5.33 

29.26 

Feb.  1 

12.9 

8.44 

320 

8.16 

119.8 

45.8 

62 

"  2 

12.7 

8.44 

310 

8.16 

73.2 

36.8 

50 

Period  2.  —  Food:  200  gm.  Meat;  120  gm.  Cracker  Meal;  50  gm.  Lard; 

30  GM. 

Bone  Ash;  400  c.c.  Water. 

Feb.  5 

12.5 

9.11 

350 

7.86 

"  6 

12.6 

9.11 

410 

8.24 

153.5 

81.5 

47 

►3.41 

15.66 

"  7 

12.6 

9.11 

450 

7.89 

114.2 

64.5 

43 

Period  3.  —  Food:  200  gm.  Meat;  160  gm.  Cracker  Meal;  30  gm.  Bone  Ash; 

450  c.c. 

Water. 

Feb. 10 
"  11 

12.4 
12.3 

9.78 
9.78 

430 
360 

7.80 
7.86 

155.7 
129.7 

59.3 
50.2 

62 
61 

1 3.37 

5.48 

the  diet  and  carbohydrate  intake  again  augmented.  Carbohydrate 
utilization  was  perfect  upon  all  diets  of  this  experiment. 

With  Dog  B  there  was  a  gradual  but  steady  loss  of  body  weight 
upon  diets  which  would  have  been  entirely  adequate  for  a  normal 
animal  of  the  same  weight.  In  the  first  period  (five  days)  a  negative 
balance  of  0.99  gm.  nitrogen  or  minus  0.19  gm.  nitrogen  per  day  was 


372 


Frank  P.  Underhill. 


obtained.  During  this  period  nitrogen  of  the  food  was  utilized  to  the 
extent  of  87  per  cent.  The  fat  utilization  was  85  per  cent,  while  that 
of  the  carbohydrate  was  perfect  —  that  is,  no  trace  of  carbohydrate 
could  be  demonstrated  in  the  faeces.    In  the  second  period  (three 


TABLE  III. 
Summary.  —  Experiment  I. 


Dog  A,  39  per  Cent 

Nitrogen. 

Periods. 

Food. 

Excreta. 

Balance. 

Urine. 

Faeces. 

Total. 

Per 
period. 

Per  day. 

1 

gm. 

21.95 

gm. 

14.07 

gm. 

2.66 

gm. 

16.73 

gm. 

+5.22 

gm. 

+  1.04 

2 

-  24.05 

16.68 

3.68 

20.36 

+3.69 

+0.74 

3 

26.15 

20.28 

3.67 

23.95 

+2.20 

+0.44 

Dog  B, 

66  per  Cent 

1 

42.20 

37.86 

5.33 

43.19 

-0.99 

-0.19 

2 

27.33 

23.99 

3.41 

27.40 

-0.07 

-0.02 

3 

19.56 

15.66 

3.37 

19.03 

+0.53 

+0.26 

days)  the  dog  was  in  almost  perfect  nitrogenous  equilibrium,  a  total 
negative  balance  of  only  0.07  gm.  or  0.02  gm.  nitrogen  per  day  being 
obtained.  During  the  period  both  carbohydrate  and  fat  intake  had 
been  markedly  increased,  nitrogen  increase  being  slight.  In  spite  of 
these  increases  the  utilization  of  the  three  components  of  the  diet  re- 
mained practically  unchanged.  The  third  period  (two  days)  reveals 
a  positive  nitrogen  balance  of  0.53  gm.  nitrogen,  or  plus  0.26  gm. 
nitrogen  per  day.  No  lard  was  fed  during  this  period,  but  carbohy- 
drate was  much  increased.  The  utilization  of  nitrogen  was  not  quite 
so  good  as  in  previous  periods.  Fat  utilization  was  greatly  dimin- 
ished.  A  portion  of  this  apparent  diminution  may  probably  be  ex- 


The  Metabolism  of  Dogs. 


373 


plained  by  the  presence  in  the  faeces  of  ether  soluble  intestinal  ex- 
cretory products  which  in  the  absence  of  truly  unutilized  food  fat 
causes  a  distortion  of  the  percentage  utilization.  Although  the  car- 
bohydrate intake  was  twice  that  of  the  first  period,  no  trace  of  sugar- 


.  TABLE  III. 
Summary    Experiment  I. 


Intestine  Resected. 

Fat  (ether  extract). 

Carbohydrate. 

Utiliza- 
tion. 

Food. 

Faeces. 

Utilization. 

Food 
(calcu- 
lated). 

Faeces. 

Utilization. 

per  cent. 

87 

gm. 

125.50 

gm. 

11.96 

per  cent. 

90 

gm. 

172 

gm. 

0 

per  cent. 

100 

84 

207.55 

27.35 

86 

273 

0 

100 

86 

29.55 

9.80 

66 

364 

0 

100 

Intestine  Resected. 

87 

199.45 

29.26 

85 

292 

0 

100 

87 

181.59 

15.66 

91 

262 

0 

100 

82 

22.36 

5.48 

75 

233 

0 

100 

yielding  substances  could  be  detected  in  the  faeces.  A  point  of  interest 
in  connection  with  the  faeces  is  the  variable  water  content. 

From  these  experiments  upon  animals  with  different  lengths  of 
intestine  put  out  of  function  shortly  after  the  operation  only  small 
differences  can  be  detected  in  the  ability  of  the  two  dogs  to  utilize 
their  food,  although  in  one  case,  Dog  A,  only  39  per  cent  of  the  en- 
tire small  intestine  was  not  functionating,  whereas  with  Dog  B  66 
per  cent  was  non-functional.  Furthermore,  notable  increases  in  fat 
intake  appeared  to  cause  little  or  no  change  in  fat,  nitrogen,  or  car- 
bohydrate utilization.  Large  increases  in  carbohydrate  did  not 
result  in  impaired  utilization.    The  carbohydrate  utilization  was 


374  Frank  P.  Underhill. 

very  much  better  than  that  of  normal  dogs  upon  practically  the  same 
diets.  For  instance,  unpublished  experiments  of  Dr.  Mary  D.  Swartz 
make  it  evident  that  only  90  to  95  per  cent  of  ingested  carbohydrate 
is  utilized  in  the  normal  dog.  Only  in  the  case  of  fat  can  the  utiliza- 
tion be  called  poor. 

TABLE  IV. 

Experiment  II.  —  Dog  A,  39  per  Cent  Intestine  Resected. 


Period  1.  —  Food:  100  gm.  Meat;  50  gm.  Cracker  Meal;  10  gm.  Lard;  10  gm. 
Bone  Ash;  150  c.c.  Water. 

Nitro- 
gen in 
food. 

Urine. 

Faeces. 

Date. 

Body 
weight. 

Vol- 

Total 

Weight 

Water 

Nitro- 

Ether 

ume. 

nitro- 
gen. 

Moist. 

Air- 
dry. 

con- 
tent. 

gen. 

extract. 

1910. 

May  25 

kilos. 

10.5 

gm. 

4.43 

c.c. 

75 

gm. 

3.77 

gm. 

23 

gm. 

13 

per  cent. 

43  • 

gm. 

gm. 

"  26 

10.4 

4.43 

80 

3.70 

32 

17 

47 

j>  1.60 

5.88 

"  27 

10.4 

4.43 

65 

3.28 

38 

25 

34 

Period  2.  —  Food:  100  gm.  Meat;  75  gm.  Cracker  Meal;  36  gm.  Lard;  10  gm. 
Bone  Ash;  230  c.c.  Water. 

May  28 

10.5 

4.88 

85 

3.44 

16 

7 

56 

"  29 

10.5 

4.88 

110 

3.51 

49 

27 

45 

"  30 

10.6 

4.88 

180 

3.42 

41 

22 

46 

>  3.44 

13.22 

"  31 

10.5 

4.88 

160 

3.28 

43 

21 

51 

June  1 

10.5 

4.88 

220 

3.33 

68 

35 

48 

Description  of  Experiment  II. 

At  the  completion  of  Experiment  I  the  dogs  were  allowed  to  run  in 
large  airy  cages  and  were  fed  upon  adequate  mixed  diets.  As  time 
progressed,  it  became  noticeable  that  Dog  B  developed  an  almost  in- 
satiable thirst.  Diarrhoea  was  persistent,  the  stools  discharged  being 
notably  clay-colored.    A  constant  but  gradual  loss  of  weight  also 


The  Metabolism  of  Dogs. 


375 


occurred.  On  the  other  hand,  Dog  A  appeared  to  be  perfectly  normal 
and  steadily  gained  in  weight.  The  rest  period  for  these  animals 
extended  from  February  12  to  May  23.  From  this  time  until  June  2, 

TABLE  V. 


Experiment  II.  —  Dog  B,  66  per  Cent  Intestine  Resected. 


Period  1.  —  Food:  200  gm.  Meat;  80  gm.  Cracker  Meal;  30  gm.  Lard;  20  gm. 
Bone  Ash;  300  c.c.  Water. 

Nitro- 
gen in 
food. 

Urine. 

Faeces. 

Date. 

Body 
weight. 

Vol- 
ume. 

Total 

Weight. 

Water 

Nitro- 
gen. 

Ether 
extract. 

nitro- 
gen. 

Moist. 

Air- 
dry. 

con- 
tent. 

1910. 

May  23 

kilos. 

10.2 

gm. 

8.50 

c.c. 

205 

gm. 

7.83 

gm. 

137 

gm. 

50 

per  cent. 

64 

gm. 

gm. 

"  24 

10.4 

8.50 

150 

6.39 

223 

67 

70 

"  25 

10.4 

8.50 

175 

7.78 

140 

59 

58 

>  6.93 

66.96 

"  26 

10.2 

8.50 

190 

7.37 

166 

60 

64 

"  27 

10.2 

8.50 

150 

7.05 

165 

64 

64 

Period  2.  —  Food:  200  gm.  Meat;  120  gm.  Cracker  Meal;  50  gm.  Lard;  30  gm. 
Bone  Ash;  400  c.c.  Water. 

May  28 

10.2 

9.22 

170 

6.99 

118 

32 

73 

"  29 

10.2 

9.22 

175 

6.96 

315 

106 

65 

"  30 

10.0 

9.22 

165 

6.96 

262 

110 

59 

>  8.63 

102.37 

"  31 

10.0 

9.22 

155 

7.26 

372 

150 

60 

June  1 

10.0 

9.22 

185 

7.56 

63 

25 

60 

two  periods  of  five  days  each  were  carried  out  upon  diets  practically 
identical  with  those  of  Experiment  1.  The  food  fed  in  these  two 
periods  corresponded  with  that  given  in  the  first  two  periods  of  Ex- 
periment 1.  Owing  to  slight  differences  of  nitrogen  content  of  the 
meat,  the  total  nitrogen  intake  varied  slightly  from  that  in  the  previ- 
ous experiment.    See  Tables  IV,  V,  and  VI. 


376 


Frank  P.  Under  hill. 


Discussion  of  Results  of  Experiment  II. 

During  the  second  period  of  observation  (Table  VI)  Dog  A  furnished 
only  positive  nitrogen  balances.  This  animal  had  39  per  cent  of  its 
small  intestine  short-circuited.    Fat  utilization  may  be  fairly  com- 


TABLE  VI. 
Summary.  —  Experiment  II. 


Dog  A,  39  per  Cent 

Nitrogen. 

Periods. 

Excreta. 

Balance. 

Food. 

Urine. 

Faeces. 

Total. 

Per  period. 

Per  day. 

1 

gm. 

13.29 

gm. 

10.75 

gm. 

1.60 

gm. 

12.35 

gm. 

+0.94 

gm. 

+0.31 

2 

24.40 

16.98 

3.44 

20.42 

+3.98 

+0.79 

Dog  B,  66  per  Cent 

1 

42.5 

36.42 

6.93 

43.35 

-0.85 

-0.17 

2 

46.1 

35.75 

8.63 

44.38 

+  1.72 

+0.34 

pared  with  that  of  a  normal  dog  on  a  mixed  diet  and  was  better  than 
in  Experiment  I  several  months  earlier.  Increase  of  fat  intake  had 
little  if  any  influence  upon  utilization  of  any  of  the  foodstuffs.  •  Car- 
bohydrate utilization  remained  perfect.  The  body  weight  of  Dog  A 
was  7.8  kilos  at  the  beginning  of  Experiment  I,  and  at  the  end  of  Ex- 
periment 2  had  increased  to  10.5  kilos  —  a  gain  of  2.7  kilos. 

It  is  at  once  apparent  from  an  inspection  of  Table  VI,  Dog  B,  that  at 
a  period  three  months  after  functional  resection  of  two  thirds  of  the 
intestine  fat  utilization  was  much  lower  than  shortly  after  the  opera- 
tion. Increasing  fat  intake  within  somewhat  narrow  limits  did  not 
markedly  impair  utilization  of  any  of  the  foodstuffs.  Carbohydrate 
utilization  was  still  perfect.  Nitrogen  utilization,  however,  was  some- 
what lowered  and  appeared  to  undergo  a  still  further  slight  diminution 


The  Metabolism  of  Dogs. 


377 


by  increase  in  fat  intake.  At  this  later  period  of  observation  the  dog 
furnished  a  slight  negative  nitrogen  balance  for  the  first  five  days  and 
a  positive  nitrogen  balance  for  the  second  period.  The  body  weight 
at  the  beginning  of  Experiment  I  was  13.3  kilos  and  at  the  end  of 
Experiment  II  was  10.0  kilos  —  a  loss  of  3.3  kilos. 


TABLE  VI. 
Summary.  —  Experiment  II. 


Intestine  Resected. 

Fat  (ether  extract). 

Carbohydrate. 

Utiliza- 
tion. 

Food. 

Faeces. 

Utilization. 

Food 
(calcu- 
lated). 

Faeces. 

Utilization. 

per  cent. 

87 

gm. 

75.48 

gm. 

5.88 

per  cent. 

92 

gm. 

109 

gm. 

0 

per  cent. 

100 

86 

206.20 

13.22 

94 

273 

0 

100 

Intestine  Resected. 

83 

201.28 

66.96 

66 

292 

0 

100 

81 

301.90 

102.37 

66 

437 

0 

100 

Description  of  Experiment  III. 

The  food  received  by  Dog  No.  12  was  the  usual  mixture  of  raw 
meat,  cracker  meal  and  lard  which  was  fed  several  days  previous  to 
the  actual  period  of  observation  and  in  sufficient  quantities  to  main- 
tain a  normal  dog  in  nitrogenous  equilibrium.  Water  was  given  ad 
libitum.  Preliminary  trials  demonstrated  the  separate  collection  of 
urine  and  faeces  to  be  almost  impracticable  owing  to  the  persistent 
diarrhoea.  To  overcome  this  obstacle  the  animal  was  fed  small  quan- 
tities of  finely  ground  agar-agar  with  the  food  —  a  procedure  which 
resulted  in  the  passage  of  stools  which  while  not  formed  were  also 
not  fluid.   The  number  of  defalcations  was  just  as  great  as  without 


378 


Frank  P.  Underhill. 


the  agar,  but  the  character  of  the  stools  was  so  entirely  altered  that 
contamination  of  the  urine  was  prevented.  In  all  probability  the 
insoluble  agar  produced  this  change  in  the  texture  of  the  faeces  by 
imbibing  the  water  from  the  intestinal  contents. 

Utilization  of  nitrogen,  fat,  and  carbohydrate.  —  In  the  first  observa- 
tion with  this  dog  it  was  planned  to  bring  about  nitrogenous  equi- 


TABLE  VII. 

Experiment  III,  Period  1.  —  Dog  No.  12,  73  per  Cent  Intestine  Resected. 


Date.  1909. 

Body  weight. 

Nitrogen  in 
food. 

Urine. 

Faeces. 

Volume. 

Total 
nitrogen. 

Ammonia 
nitrogen. 

Indican 
fehlings 
sol.  =  100. 

We 

.22 
'o 

3 

ight. 

•i  £ 

Water 
content. 

Nitrogen. 

Ether 
extract. 

Jan. 

kilos. 

gm. 

c.c. 

gm. 

gm, 

gm. 

gm. 

per  cent. 

gm. 

gm. 

14 

7.0 

8.92 

130 

6.48 

0.34 

10 

210 

36 

83 

15 

7.0 

8.24 

125 

6.00 

0.55 

10 

232 

37 

84 

^8.70 

18.70 

16 

7.0 

8.24 

220 

8.40 

0.70 

12 

67 

79 

14 

82 

17 

6.8 

8.24 

210 

6.86 

12 

7 

librium  as  nearly  as  possible  and  then  to  determine  the  utilization  of 
the  different  foodstuffs.  To  accomplish  this  purpose  a  few  days 
previous  to  the  real  observation  the  dog  received  a  diet  consisting  of 
200  gm.  meat,  120  gm.  cracker  meal  and  10  gm.  lard  containing  8.92 
gm.  nitrogen  and  furnishing  approximately  775  calories,  or  1.27  gm. 
nitrogen  and  no  calories  per  kilo  body  weight,  amounts  which  would 
be  far  in  excess  of  the  requirements  for  a  normal  dog  of  this  size. 
This  diet  was  continued  through  the  first  day  (January  14)  of  obser- 
vation and  was  then  reduced,  since  the  animal  appeared  to  have 
difficulty  for  the  first  time  in  devouring  these  large  amounts  of  food. 
The  new  diet  contained  200  gm.  meat,  80  gm.  cracker  meal,  10  gm. 
lard,  and  10  gm.  agar.  The  nitrogen  content  amounted  to  8.24  gm. 
This  diet  was  eaten  readily. 

During  the  four  days  of  observation,  the  results  of  which  may  be 
seen  in  Tables  VII  and  IX,  it  is  apparent  that  the  dog  was  not  in  a 
condition  of  nitrogenous  equilibrium  in  spite  of  the  previous  ingestion 
of  large  quantities  of  food,  the  nitrogenous  balance  for  the  four  days 


The  Metabolism  of  Dogs. 


379 


being  minus  2.80  gm.,  or  minus  0.7  gm.  nitrogen  per  day.  Turning 
to  the  utilization  of  nitrogen  and  fat,  it  may  be  observed  that  both 
were  poor,  nitrogen  being  utilized  to  the  extent  of  only  74  per  cent  fat 

TABLE  VIII. 


Experiment  III,  Periods  2  and  3. —  Dog  No.  12,  73  per  Cent  Intestine 

Resected. 


Period  2.  —  Food  per  Day:  100  gm.  Meat;  25  gm.  Gelatin;  80  gm.  Cracker  Meal; 
10  gm.  Lard;  10  gm.  Agar- agar.   Total  Nitrogen  =  8.24  gm. 

Urine. 

Faeces. 

Date. 

Vol- 

Total 
nitro- 
gen. 

Indican 
fehl- 

Weight. 

Water 

Nitro- 

Ether 

ume. 

ings 
solution 
=  100. 

Moist. 

Air-dry. 

con- 
tent. 

gen. 

extract. 

1909. 

Jan.  22 

c.c. 

120 

gm. 

6.38 

7 

gm. 

112 

gm. 

20 

per  cent. 

82 

gm. 

gm. 

"  23 

160 

6.57 

9 

48 

"  24 

175 

7.49 

8 

201 

51 

80 

>  7.19 

15.80 

"  25 

250 

10.10 

8 

86 

18 

80 

"  26 

160 

6.72 

10 

159 

24 

84 

Period  3.  —  Food  per  Day:  50  gm.  Gelatin;  80  gm.  Cracker  Meal;  10  gm.  Lard; 
10  gm.  Agar-agar.   Total  Nitrogen  =  8.96  gm. 

Jan.  27 

450 

8.88 

11 

1 

146 

26 

82 

"  28 
"  29 

430 
170 

7.44 
7.58 

8 
8 

161 

35 
29 

82 

>  8.91 

24.90 

"  30 

160 

7.51 

9 

32 

to  the  extent  of  72  per  cent.  These  figures  agree  well  with  those  of 
Erlanger  and  Hewlett.  In  spite  of  the  extremely  foul  odor  of  the 
faeces  the  quantity  of  indican  eliminated  through  the  urine  was  not 
excessive.  The  figures  for  ammonia  nitrogen  are  high  when  com- 
pared to  the  output  of  the  normal  dog.  Another  point  of  interest 
was  the  rather  large  percentage  of  water  contained  in  the  faeces  of 
this  animal.   Normally  the  water  content  of  the  air-dry  faeces  rarely 


38o 


Frank  P.  Under  hill. 


exceeds  75  per  cent,  whereas  those  passed  by  this  animal  had  a  water 
content  of  80  to  85  per  cent  (see  also  Table  VIII).  It  is  not  unlikely 
that  the  water  content  of  the  faeces  may  have  been  increased  by  the 
presence  of  agar,  which  would  imbibe  a  certain  quantity  of  water  and 
prevent  its  absorption.  It  is  hardly  probable,  however,  that  this 
is  the  sole  explanation  since  the  fluidity  of  the  faeces  passed  when 

TABLE  IX. 


Summary.  —  Experiment  III,  Dog  No.  12,  73  per  cent  Intestine  Resected. 


Peri- 
ods. 

Nitrogen. 

Fat  (ether  extract). 

Food. 

Excreta. 

Balance. 

Utili- 
zation. 

Food. 

Faeces. 

Utili- 
za- 
tion. 

Urine. 

Faeces. 

Total. 

Per 
period. 

Per 
day. 

gm. 

gm. 

gm. 

gm. 

gm. 

gm. 

per  cent. 

mg. 

gm. 

per  cent. 

1 

33.64 

27.74 

8.70 

36.44 

-2.80 

-0.70 

74 

67.60 

18.70 

72 

2 

41.20 

37  26 

7.19 

44.45 

-3.25 

-0.64 

82 

96.50 

15.80 

84 

3 

35.84 

31.41 

8.91 

40.32 

-4.48 

-1.12 

75 

59.20 

24.90 

58 

no  agar  was  given  was  such  that  a  large  water  content  was  obvious. 
By  varying  the  amount  of  carbohydrate  even  up  to  four  times  the 
requirement  for  a  normal  dog  of  the  same  weight,  no  change  in  the 
utilization  of  this  foodstuff  could  be  observed.  It  has  been  demon- 
strated repeatedly  in  this  laboratory  that  a  diet  sufficient  for  a  normal 
dog  of  this  weight  may  consist  of  200  gm.  meat,  30  gm.  cracker  meal, 
and  25  gm.  lard.  Dog  No.  12  received  approximately  this  diet,  then 
the  cracker  meal  was  increased  to  80  gm.  and  the  lard  reduced  to 
10  gm.  Finally,  the  cracker  meal  was  still  further  increased  to  120 
gm.  In  all  cases  carbohydrate  utilization  was  complete.10  In  these 
experiments  designed  to  test  carbohydrate  utilization  agar  was  not 
given  with  the  food  for  obvious  reasons. 

The  Influence  of  Gelatin  Feeding  upon  the  Elimination  of 

Urinary  Indican. 

In  a  previous  communication  11  it  was  demonstrated  that  the  re- 
placement of  meat  by  gelatin  in  a  mixed  diet  results  in  a  diminution 

10  Cf.  Flint:  hoc.  ciL         11  Underhill:  This  journal,  1904-1905,  xii,  p.  176. 


The  Metabolism  of  Dogs. 


381 


in  the  excretion  of  indican  in  the  urine  of  the  dog.  This  observation 
is  in  accord  with  the  now  well-established  origin  of  indican.  In  Table 
VIII  (Periods  2  and  3)  are  given  the  results  of  observations  made 
with  a  view  of  determining  whether  a  dog  with  a  short-circuited  in- 
testine would  behave  in  a  manner  similar  to  a  normal  animal  when  a 
portion  or  all  the  meat  of  the  diet  was  replaced  by  gelatin.  It  is  ob- 
vious from  these  figures  that  the  substitution  of  gelatin  for  meat  was 
without  significant  influence  upon  urinary  indican  elimination.  With 
this  animal  only  negative  nitrogen  balances  were  obtained.  In  the 
second  gelatin  period  nitrogen  utilization  amounted  to  75  per  cent, 
which  is  comparable  to  the  utilization  obtaining  when  meat  was  fed 
(see  Table  IX,  Period  1).  The  fat  utilization  in  the  first  gelatin 
period  (see  Table  IX,  Period  2)  when  some  meat  was  fed  was  much 
better  than  when  the  meat  was  entirely  replaced  by  gelatin.  In  the 
first  instance  utilization  amounted  to  84  per  cent;  in  the  second 
gelatin  period  (see  Table  IX,  Period  3)  only  58  per  cent  of  the  fat  was 
utilized.  It  would  appear  that  meat  in  the  diet  of  this  animal  had  a 
tendency  to  aid  fat  utilization. 


Summary. 

From  the  foregoing  observations  it  is  apparent  that  as  much  as 
39  per  cent  of  the  small  intestine  of  a  dog  may  be  short-circuited 
without  causing  significant  detrimental  changes  in  the  utilization  of 
the  various  foodstuffs,  and  the  animal  may  gain  in  weight.  This 
statement  is  equally  true  when  observations  are  made  either  at  a 
period  shortly  after  operation  or  at  a  period  several  months  later. 

When  as  much  as  66  per  cent  of  the  small  intestine  has  been  func- 
tionally resected,  the  nutritive  condition  of  the  animal  presents  an 
entirely  different  aspect.  Under  these  conditions  fat  utilization  is 
particularly  decreased  and  the  dog  displays  a  decided  tendency  to 
furnish  negative  nitrogen  balances.  A  small  though  steady  loss  of 
weight  is  especially  noticeable.  Food  utilization  is  in  general  ap- 
parently much  better  immediately  after  the  operation  than  at  a  later 
period.  In  neither  animal  did  a  material  increase  in  fat  intake  cause 
significant  change  in  the  utilization  of  this  or  other  foodstuff. 

When  about  three  quarters  of  the  small  intestine  of  the  dog  has 
been  short-circuited,  food  utilization  for  the  most  part  is  seriously 


382 


Frank  P.  Underbill. 


iir  paired,  at  least  at  a  period  several  months  after  the  operation.  This 
is  particularly  true  for  fat  utilization.  Indican  elimination  through 
the  urine  is  not  materially  altered  under  these  conditions  by  replace- 
ment of  meat  in  the  diet  with  gelatin,  an  observation  directly  opposed 
to  that  obtained  with  the  normal  dog. 

The  animal  with  a  short-circuited  intestine  displays  a  greater 
ability  to  utilize  carbohydrate  than  does  the  normal  dog.  Even 
though  the  carbohydrate  intake  may  be  much,  in  one  case  several 
times,  greater  than  the  normal  animal  requires,  carbohydrate  utiliza- 
tion is  complete  whether  the  test  is  made  shortly  after  the  operation 
or  months  later.  This  observation  may  prove  of  practical  importance 
in  the  dietary  treatment  of  the  human  subject  who  has  undergone 
extensive  intestinal  resection. 


Reprinted  from  the  American  Journal  of  Physiology. 
Vol.   XXVIII.  —  August   1,    1911. —  No.  V. 


ACAPNIA  AND  GLYCOSURIA. 

By  YANDELL  HENDERSON  and  FRANK  P.  UNDERHILL. 

[From  the  Physiological  Laboratory,  Yale  Medical  School,  and  the  Sheffield  Laboratory  of 
Physiological  Chemistry,  Yale  University,  New  Haven,  Connecticut.] 

I.  The  Point  of  View. 

GLYCOSURIA  is  induced  by  an  extraordinarily  large  number  of 
widely  differing  conditions.  This  fact  indicates  that  it  is  the 
result  of  a  disturbance  of  a  complex  balance  involving  many  factors. 
With  the  exception  of  phloridzin  diabetes,  practically  all  forms  of 
glycosuria,  whether  experimental  or  clinical,  are  the  result  of  a  hyper- 
glycemia. The  excess  of  sugar  in  the  blood  is  in  turn,  not  a  funda- 
mental phenomenon,  but  the  expression  of  diminished  ability  on  the 
part  of  the  tissues  to  utilize  dextrose.  Thus  the  seat  of  the  complex 
equilibrium  is  in  the  cells,  not  the  fluids,  of  the  body. 

The  problem  of  the  normal  glycogenic  function  and  of  experimental 
diabetes  consists  in  determining  the  various  factors  in  this  equilibrium 
and  denning  their  relations.  The  problem  of  any  clinical  form  of 
glycosuria  lies  in  discovering  which  particular  factor  or  set  of  factors 
is  altered  from  its  normal  strength,  and  how. 

One  of  the  most  important  of  the  sets  of  factors  involved  in  the 
normal  balance  is  the  internal  or  tissue  respiration.  In  his  investiga- 
tion^ on  various  forms  of  experimental  diabetes,  Underhill 1  noted  the 
casual  connection  of  these  conditions  with  disturbances  of  breathing. 
Araki 2  has  shown  that  an  insufficient  supply  of  oxygen  to  the  cells 
(e.  g.y  in  CO  poisoning)  results  in  a  notable  glycosuria.  Macleod  3 
reached  the  conclusion  that  in  asphyxia  it  is  the  excess  of  CO2  and  not 
the  deficiency  of  oxygen,  which  stimulates  hepatic  glycogenolysis.  The 

1  Underhill,  F.  P.:  Journal  of  biological  chemistry,  1905,  i,  p.  113. 

2  Araki:  Zeitschrift  fur  physiologische  Chemie,  1891,  xv,  p.  351. 

3  Macleod,  J.  J.  R.:  This  journal,  1909,  xxiii,  p.  302. 

275 


276         Yandell  Henderson  and  Frank  P.  Under  hill. 


work  of  Edie,4  and  of  Edie,  Moore,  and  Roaf 5  seems  to  demonstrate 
that  an  excess  of  CO2  in  the  air  breathed  apart  from  oxygen  deficiency 
may  result  in  glycosuria. 

We  proposed  to  ourselves  the  question:  Will  acapnia  also  upset  the 
capacity  of  the  tissues  to  hold  sugar?  The  observations  to  be  here 
reported  indicate  that  such  is  the  case.  One  may  bring  a  see-saw  to 
the  ground  as  well  by  lessening  as  by  increasing  the  weight  on  one 
end.  Our  observations  indicate  also  that  acapnia  is  a  concomitant  of 
some  forms  of  experimental  glycosuria  in  which  Edie,  Moore,  and 
Roaf  have  held  that  hypercapnia  must  occur. 

Glycosuria  is  known  to  follow  violent  emotion  in  human  subjects 
and  to  occur  in  cats  which  rage  at  being  tied.6  Henderson  7  has 
pointed  out  that  the  stormy  breathing  .of  anger  involves  excessive 
pulmonary  ventilation.  He  has  likewise  shown  that  ether  anaesthesia 
in  dogs  without  morphin  nearly  always  involves  a  greater  or  less 
degree  of  acapnia,  never  hypercapnia.  It  is  noteworthy  that  a  tem- 
porary glycosuria  is  a  frequent  sequel  of  ether  anaesthesia.  Prolonged 
ether  excitement  may  reduce  the  C02  content  of  the  blood  to  half  the 
normal  amount,  and  result  fatally.8  These  effects,  including  the  glyco- 
suria, are  solely  due  to  the  excessive  breathing  induced  by  ether  excite- 
ment.9 We  have  never  found  sugar  in  the  urine  of  dogs  which  had 
been  brought  into  deep  ether  anaesthesia  quietly.  We  believe  that 
both  emotional  glycosuria  and  polyuria  are  frequently  the  result  of 
acapnia. 

Acetone  has  an  influence  even  more  powerful  than  ether  in  excit- 
ing respiration  to  excessive  activity.  When  administered  to  normal 
animals  it  has  been  shown  to  produce  glycosuria.10  The  close  asso- 
ciation of  this  substance  with  the  most  important  clinical  form  of 

4  Edie:  Biochemical  journal,  1906,  i,  p.  455. 

5  Edie,  Moore,  and  Roaf:  Biochemical  journal,  1911,  v,  p.  325. 

6  Bohm  and  Hoffmann:  Archiv  fur  experimentelle  Pathologie  und  Pharma- 
kologie,  1878,  viii,  p.  271. 

7  Henderson,  Y.:  This  journal,  1910,  xxv,  p.  311. 

8  Henderson,  Y.:  This  journal,  1910,  xxvi,  p.  280. 

9  Underhill:  hoc.  cit. 

10  Albertoni:  Archiv  fur  experimentelle  Pathologie  und  Pharmakologie,  1884, 
xviii,  p.  218;  v.  Jaksch:  Zeitschrift  fiir  klinische  Medizin,  1886,  x,  p.  362; 
Ruschhaupt:  Archiv  fiir  experimentelle  Pathologie  und  Pharmakologie,  1900 
xliv,  p.  127;  Muller:  Ibid.,  1901,  xlvi,  p.  67. 


Acapnia  and  Glycosuria. 


277 


glycosuria  raises  the  question  to  what  extent  acapnia  may  occur,  and 
what  its  significance  may  be,  in  diabetes  mellitus.  Acetone  is  prob- 
ably a  factor  in  that  "  nervousness  "  of  diabetics  which  is  in  part  the 
subjective  expression  of  their  proneness  to  hyperpncea. 

In  diabetic  coma  a  degree  of  acapnia  has  been  observed  more  in- 
tense than  in  any  other  known  condition.  Magnus  Levy  11  found  only 
3.3  per  cent  of  CO2  in  the  blood  just  before  the  death  of  a  patient,  — 
less  than  one  tenth  of  the  normal.  It  is  generally  assumed  that  this 
is  not  a  true  acapnia  such  as  results  from  excessive  breathing,  but  that 
it  is  merely  a  pseudo-acapnia  due  to  the  expulsion  of  CO2  from  the 
bicarbonates  of  the  blood  by  organic  acids.  This  view  involves,  how- 
ever, a  large  measure  of  assumption.  To  prove  it  would  require  simul- 
taneous determinations  of  the  CO2  tension,  C02  content,  and  hydro- 
+ 

gen  ion  concentration  (H)  of  the  blood.  No  one  has  yet  performed  this 
difficult  task.12 

The  line  of  reasoning  usually  adopted  starts  from  the  fact  that 

(1)  Acids  are  formed  in  the  tissues  and  pass  into  the  blood  in  acidosis. 

(2)  Acids  liberate  CO2  from  bicarbonates.  And  (3)  during  acidosis 
the  CO2  content  of  the  blood  is  greatly  diminished.  From  these  three 
facts  the  conclusions  are  drawn  that:  (1)  In  acidosis  the  acids  are 
produced  in  an  amount  sufficient  to  explain  the  low  content  of  CO2 
wholly  by  expulsion.    (2)  The  capacity  of  the  blood  to  carry  CO2  is 

destroyed  or  greatly  diminished.  And  (3)  the  acidity  or  (H)  is  in- 
creased.  The  facts  scarcely  warrant  any  of  these  conclusions. 

Beddard,  Pembrey,  and  Spriggs 13  found  that  diabetic  blood  is 
quite  capable  of  taking  up  large  quantities  of  CO2  when  exposed  to 
the  gas  in  vitro.  Evidently  the  alkalies  of  the  blood  even  in  acute 
diabetic  acapnia  are  far  from  being  completely  neutralized  by  oxybu- 
tyric  and  other  organic  acids.  These  investigators  have  also  recorded 
the  exceedingly  important  observation  that  during  diabetic  coma  the 
CO2  tension  of  the  alveolar  air  of  the  lungs  is  far  below  normal.  Thus 
they  have  demonstrated  that  the  acapnia  incident  to  acidosis  is  mainly 

11  Magnus  Levy:  Archiv  fur  experimentelle  Pathologie  und  Pharmakologie, 
1901,  xlv,  p.  389. 

12  For  methods  see  Krogh,  Skandinavisches  Archiv  fur  Physiologie,  1908,  xx, 
p.  279;  Hasselbalch:  Biochemische  Zeitschrift,  1910,  xxx,  p.  317.  Michaelis 
and  Rona:  Ibid.,  1909,  xviii,  p.  317. 

13  Beddard,  Pembrey  and  Spriggs:  Journal  of  physiology,  1904,  xxxi,  p.  xliv. 


278        Yandell  Henderson  and  Frank  P.  Underbill. 


a  true  low  tension  acapnia  due  to  hyperpncea.    From  this  fact  does 

+ 

it  not  follow  that  the  (H)  of  diabetic  blood  is  below  normal,  and  that 
the  blood  during  acidosis  is  less  "acid  "  than  normally,  in  spite  of  the 
neutralization  of  a  part  of  its  alkali  bicarbonates  by  strong  organic 
acids?  The  pulmonary  ventilation  of  this  condition  is  so  excessive 
that  it  exhales  not  only  the  CO2  liberated  from  combination  with 
alkalies,  but  also  a  part  of  that  amount  which  is  normally  held  in  the 
blood  in  simple  solution. 

It  is  preeminently  that  portion  of  carbonic  acid  which  is  merely 

dissolved,  uncombined  with  alkalies,  which  according  to  the  formulae 

+ 

of  L.  J.  Henderson 14  determines  the  (H)  of  the  blood.  This  portion 
obeys  the  law  of  Henry.  At  equal  partial  pressures  of  CO2  in  the 
lungs  the  carbonic  acid  dissolved  as  such  will  be  the  same  in  normal 
and  in  acidosis  blood.  Whichever  is  exposed  to  the  lower  gas  pres- 
sure will  have  the  less  CO2  in  simple  solution,  and  to  this  extent  the 
+ 

lower  (H).  As  L.  J.  Henderson  has  pointed  out  in  his  discussion  of 
the  neutrality  equilibrium  of  the  body,  lactic,  oxybutyric,  and  the 
other  diabetic  acids  when  thrown  into  the  blood  in  moderate  amount 
are  almost  completely  neutralized  by  alkalies  from  bicarbonates  and 

phosphates.    Short  of  so  intense  an  acidosis  as  to  absorb  practically 

+ 

all  of  the  alkalies  of  the  bicarbonates,  the  (H)  continues  to  depend 
principally  just  as  under  normal  conditions  upon  the  pressure  of  CO2 
in  the  alveolar  air  of  the  lungs.  In  acute  acidosis  this  pressure  is  much 
below  normal. 

Furthermore  the  body  aids  itself  in  neutralizing  the  diabetic  acids 
by  an  abnormally  large  amount  of  nitrogen  in  the  form  of  ammonia  in 
the  blood  and  urine.  The  process  of  urea  formation  is,  like  glycoge- 
nolysis,  an  equilibrium  of  many  factors,  of  which  carbonic  acid  is  one.15 

From  the  foregoing  considerations  it  is  evident  that  the  excessive 
respiration  associated  with  many  forms  of  glycosuria  cannot  be  due  to 
an  unusually  high  pressure  of  CO2  in  the  blood.  Neither  can  it  be  due 
to  a  high  acidity.  It  is  almost  certainly  due  to  the  acidosis  sub- 
stances, of  which  a  certain  quantity  is  present  in  the  blood  even  in 
health;  but  in  our  opinion  it  is  probably  the  ethereal  qualities  of 

14  Henderson,  L.  J.:  Ergebnisse  der  Physiologie,  1909,  viii,  p.  254. 

15  Macleod  and  Haskins:  Journal  of  biological  chemistry,  1906,  i,  p.  319. 


Acapnia  and  Glycosuria. 


279 


these  bodies  and  not  their  acidic  influence  which,  when  added  to  the 

C02  remaining  in  the  blood,  excites  the  respiratory  centre  to  maintain 

+ 

the  tension  of  this  gas,  and  with  it  the  (H),  below  normal.  That  such  a 

conception  is  a  possibility  is  proven  by  Henderson's  observations  that 

a  fatal  acapnia  may  be  induced  by  means  of  ethyl  ether,  and  by  the 

fact  that  acetone  is  an  even  more  powerful  respiratory  excitant. 

A  number  of  investigators  have  noted  that  the  intravenous  infusion 

of  acids  exerts  a  very  brief  influence  upon  breathing.   The  acids  are 

immediately  neutralized  into  their  salts;  the  CO2  thus  displaced  is 
+ 

exhaled;  and  the  (H)  depends  as  before  upon  the  carbonic  acid  in 
solution. 

The  effect  of  the  administration  of  alkalies  in  diminishing  the 
dyspnoea  during  diabetic  coma  can  in  like  manner  be  reconciled  with 

our  view.16    If  the  activity  of  the  respiratory  centre  is  due  to  the  sum- 

+ 

mated  influence  of  the  ethereal  acidosis  bodies  and  the  C02  or  (H)  in 
the  blood,  then  diminishing  the  second  term  in  this  sum  must  reduce 
their  combined  stimulating  influence  in  precisely  the  same  manner  as 
that  by  which  alkalies  lessen  the  breathing  of  a  normal  subject.  It 

matters  not  whether  the  influence  of  carbonic  acid  upon  the  respiratory 

+ 

centre  be  regarded  as  depending  upon  (H)  (Winterstein  17)  or  whether, 
as  appears  to  us  more  probable,  this  centre  is  specifically  irritable  to 
CO2  apart  from  its  acidity. 

With  such  tentative  conceptions  as  these  in  mind  we  undertook  the 
experiments  here  to  be  reported.  They  show  that  acapnia  is  in  fact  a 
constant  accompaniment  of  all  the  forms  of  experimental  glycosuria 
studied  by  us.  Upon  the  more  important  point,  whether  or  not  prevent- 
ing the  development  of  acapnia  will  prevent  glycosuria,  the  evidence 
which  we  have  to  report  is  incomplete.  Apparently  in  some  of  these 
conditions  such  is  the  case,  in  others  not.  We  have  found  it  far  more 
difficult  than  we  had  expected  to  administer  an  atmosphere  so  rich  in 
CO2  as  to  prevent  acapnia.  As  a  suggestion  to  others  who  may  in 
future  work  on  this  or  related  topics,  and  as  a  criticism  upon  much  of 
the  work  previously  published  we  would  point  out  that  absolutely  the 
only  way  to  find  out  whether  an  animal  under  any  experimental  con- 
ditions is  acapnic  or  hypercapnic  is  by  means  of  analyses  either  of  the 

16  Cf.  Labbe  and  Violle:  Presse  medicale,  Paris,  1911,  xix,  p.  292. 

17  Winterstein,  H. :  Archiv  fur  die  gesammte  Physiologie,  191 1,  cxxxviii,  p.  167. 


280 


Yandell  Henderson  and  Frank  P.  Underbill. 


blood  or  the  alveolar  air.  In  many  of  our  own  experiments  we  should 
have  supposed  the  C02  content  to  be  above  normal  when  analyses 
actually  showed  it  to  be  much  below.  Furthermore,  as  a  commentary 
upon  the  theorizing  of  previous  workers,  it  must  always  be  remem- 
bered that  the  only  certain  criterion  of  whether  the  tissues  of  the 
body  are  adequately  supplied  with  oxygen  is  the  demonstration  of  a 
considerable  quantity  of  this  gas  in  the  venous  blood.  The  arterial  blood 
may  be  saturated  and  arterial  pressure  high,  yet  if  the  blood  stream  is 
small  the  supply  of  oxygen  will  be  insufficient  for  internal  respiratory 
needs  and  the  resulting  hyperglycemia  may  be  mistakenly  assigned 
to  other  than  its  true  cause. 

Most  of  the  forms  of  acapnia  thus  far  described  by  Henderson  18 
have  been  the  result  of  excessive  breathing  induced  by  external  influ- 
ences upon  afferent  nerves.  We  would  point  out  that  perversions  of 
metabolism  (e.  g.,  in  diabetes  and  in  fever)  may  produce  substances 
capable  of  exciting  the  respiratory  centre  to  abnormal  activity.  Thus 
acapnia  may  be  a  factor  in  the  pathologic  physiology  of  the  diseases  of 
internal  medicine  no  less  than  in  those  of  surgery. 

II.  Peptone  Glycosuria. 

The  functional  disturbances  induced  by  intravenous  injection  of 
proteoses  have  been  studied  by  a  long  series  of  investigators.  In  addi- 
tion to  the  effects  of  such  injections  upon  the  coagulability  of  the 
blood  and  upon  arterial  pressure,  one  of  the  most  obvious  results  of 
such  injections  is  that  upon  respiration.  The  animal  exhibits  great 
excitement  with  struggles,  cries,  and  labored  breathing. 

It  was  found  by  Lahousse  19  in  Ludwig's  laboratory  that  in  dogs  in 
which  the  arterial  blood  contained  33.4  to  39.5  volumes  per  cent  of 
CO2  prior  to  peptone  injection,  a  sample  of  blood  in  four  minutes  after 
the  injection  (i.  e.,  when  the  condition  of  shock  had  fully  developed) 
contained  only  16.3  to  22.4  per  cent  C02.  The  oxygen  content  of  the 
blood  was  slightly  increased.  The  indications  were  against  the  view 
that  the  tissue  oxidations  were  diminished.  Lahousse  observed  that 
the  acapnia  lasted  as  long  as  the  condition  of  shock,  and  that  the  CO2 
content  of  the  blood  rose  again  toward  normal  as  recovery  progressed. 

18  Henderson,  Y. :  This  journal,  1910,  xxvi,  p.  280. 

19  Lahousse:  Archiv  fur  Physiologie,  1889,  p.  77. 


Acapnia  and  Glycosuria. 


281 


Glycosuria  has  never,  so  far  as  we  can  find,  been  observed  as  a  re- 
sult of  peptone  injection.  The  consideration  advanced  in  the  previous 
section  led  us,  however,  to  expect  that  it  must  occur.  Our  observations 
confirm  those  of  Lahousse  and  in  addition  verify  our  expectation  that 
glycosuria  or  at  least  hyperglycemia  is  a  sequel  of  peptone  shock,  and 
that  it  is  due  to  acapnia.20 

They  demonstrate  that  the  excessive  respiration  results  in  acapnia 
and  is  succeeded  by  a  period  of  subnormal  breathing,  during  which  the 
CO2  content  of  the  blood  rapidly  returns  to  normal.  Glycosuria  fol- 
lows. If,  however,  during  the  period  of  excitement  excessive  pul- 
monary ventilation  is  prevented  by  compelling  the  subject  to  breathe 
through  a  long  tube  attached  to  the  trachea,  neither  glycosuria  nor 
hypercglyaemia  occur.  Our  experiments  further  indicate  that  the 
lowering  of  arterial  pressure  following  peptone  injection  is  not  a 
factor  in  the  disturbance  of  sugar  control. 

Dog  of  4  kilos  under  ether.  Urine  free  from  sugar.  At  11.07  m~ 
jected  2.5  gm.  Witte  peptone  in  35  c.c.  water  into  jugular  vein.  Vio- 
lent hyperpncea  for  a  few  minutes.  At  1 2 .00  the  animal  had  practically 
recovered.  Urine  was  full  of  sugar.  The  NH4  fraction  of  its  nitrogen 
was  13.5  per  cent. 

Cat  of  2.1  kilos  under  ether.  At  11.30  injected  1.0  gm.  peptone  in 
20  c.c.  water  into  jugular  vein.  Hyperpncea  resulted.  -At  12.21 
urine  was  full  of  sugar. 


Experiment  of  Oct.  29,  igoy.  —  Dog  of  10  kilos.    Ether.    Injected  into 
femoral  vein  3  gm.  Witte  peptone  in  40  c.c.  water.  Vigorous  hyperpncea. 


Time. 

Arterial  gases. 

Blood 
sugar. 

Urinary. 

Notes. 

o3 

co2 

per  cent. 

NH  :  N. 

9.50 

18.0 

30.0 

0.16 

6.9 

10.08 

Peptone  injected. 

10.12 

20.5 

21.0 

0.12 

10.55 

9.0 

Urine  full  of  sugar. 

11.30 

20.0 

26.0 

0.26 

20  Henderson  and  Underhill:  Proceedings  of  the  Society  for  Experimental 
Biology  and  Medicine,  191 1,  viii,  p.  80. 


282 


Yandell  Henderson  and  Frank  P.  Underbill. 


Experiment  of  Dec.  5,  igoy.  —  Dog,  23  kilos.   Ether.   Injected  12  gm.  pep- 
tone in  100  c.c.  water.    Hyperpncea  resulted. 


Time. 

Arterial  gases. 

Venous 
gases. 

Arterial 
pressure. 

T>1  _  _  J 

Jdiooq 
sugar. 

Notes. 

o2 

C02 

o2 

C02 

MM.Hg. 

% 

12.40 

24.0 

40.3 

16.6 

45.6 

145 

0.15 

Urine  free  from  sugar. 

12.44 

55 

Peptone  injected.  Hy- 
perpncea. 

1.00 

18.7 

30.6 

6.6 

39.0 

45 

1.40 

23.0 

16.0 

16.4 

33.4 

3.12 

25.0 

12.6 

38.8 

95 

0.27 

Urine  free  from  sugar. 

Experiment  of  Dec.  14,  igoy. —  Dog,  11.5  kilos.  At  first  chloroformed;  then 
ether.  Injected  6.0  gm.  peptone.  Hyperpncea  resulted.  Tube  at- 
tached to  trachea  2  m.  long  with  13  mm.  above. 


Time. 

Arterial  gases. 

Venous  gases. 

Arterial 
pressure. 
mm.Hg 

% 

Blood 
sugar. 

Notes. 

o2 

C02 

o2 

C02 

11.45 

150 

11.46 

50 

Peptone  injected. 

11.55 

24.1 

32.7 

13.6 

45.8 

Blood  does  not  clot. 

12.00 

Tube  attached  to  trachea. 

12.44 

75 

Tongue  pink;  breathing 

deeply. 

1.12 

23.9 

38.5 

14.3 

51.3 

85 

Blood  does  not  clot. 

2.15 

0.17 

Urine  free  from  sugar. 

III.  Piqure  Diabetes. 

The  Bernard  puncture  of  the  floor  of  the  fourth  ventricle  in  a  rabbit 
is  usually  followed  immediately  by  hyperpncea.  This  excessive  breath- 
ing continues  for  five  or  ten  minutes,  and  is  succeeded  by  a  compen- 


Acapnia  and  Glycosuria. 


283 


satory  period  of  subnormal  breathing.  From  half  an  hour  to  an  hour 
after  the  operation  polyuria  and  glycosuria  set  in.  These  conditions 
usually  last  for  four  or  five  hours  and  then  pass  off,  leaving  the  animal 
in  apparently  normal  condition. 

The  experiments  detailed  below  show  that  a  marked  acapnia  results 
from  this  hyperpncea.  Lahousse  21  has  observed  that  during  the  time 
which  we  have  denominated  above  as  the  compensatory  period  the 
respiratory  quotient  is  low.  He  regards  this  fact  as  indicating  an  al- 
teration in  the  nature  of  the  substances  undergoing  combustion  in  the 
tissues.  A  simpler  explanation  appears  to  us  to  be  that,  owing  to  the 
acapnia,  breathing  in  this  period  is  subnormal.  It  is  sufficient  to 
supply  all  the  oxygen  the  tissues  need,  but  diminishes  the  elimination 
and  thus  allows  reaccumulation  of  CO2.  During  any  period  when  an 
animal  is  diminishing  its  store  of  CO2  by  hyperpncea  or  reaccumulat- 
ing  its  normal  stock  by  subnormal  breathing  the  respiratory  quotient 
would  be  correspondingly  increased  or  diminished.  If  at  the  same  time 
the  quantity  of  oxygen  absorbed  by  the  lungs  continues  at  an  approxi- 
mately normal  rate,  the  respiratory  quotient  may  afford  an  entirely 
misleading  indication  of  the  nature  of  the  substances  undergoing 
combustion. 

Another  object  of  our  experiments  was  to  determine  whether  the  pre- 
vention of  acapnia  would  likewise  prevent  glycosuria  after  piqure. 
For  this  purpose  the  rabbits  were  placed  immediately  after  the  opera- 
tion in  a  small  chamber  supplied  with  an  ample  stream  of  air  to  which 
known  quantities  of  C02  had  been  added.  To  our  surprise  we  found  it 
by  no  means  easy  to  prevent  acapnia.  With  small  percentages  of  C02 
(5  per  cent  or  less)  the  hyperpncea  induced  by  piqure  is  sufficient  to 
result  in  a  marked  reduction  of  the  body's  store  of  C02.  Owing  to 
the  loss  of  time  involved  in  learning  this,  we  were  obliged,  by  pres- 
sure of  other  work,  to  leave  many  points  undecided.  Three  years 
have  passed  without  our  finding  an  opportunity  to  resume  the  in- 
vestigation. In  the  recent  paper  of  Edie,  Moore,  and  Roaf 22  it  is  argued 
that  hypercapnia  is  a  factor  in  many  forms  of  experimental  diabetes. 
Our  data  demonstrate  that  such  is  not  the  case  in  any  of  the  forms  of 
experimental  glycosuria  studied  by  us. 

21  Lahousse:  Archives  mternationales  de  physiologie,  1907,  v,  p.  105.  See  also 
La  Franca:  Zeitschrift  fur  experimentelle  Pathologie  und  Therapie,  1910,  vi, 
p.  1. 

22  Edie,  Moore,  and  Roaf:  hoc.  cit. 


284 


Yandell  Henderson  and  Frank  P.  Underhill. 


The  data  here  tabulated  show  that  the  prevention  of  acapnia  obviates 
polyuria  and  probably  to  some  extent  delays  and  diminishes,  but  does 
not  entirely  prevent,  the  excretion  of  sugar  after  piqure.  It  is  note- 
worthy that  in  one  of  our  control  experiments  no  apparent  disturb- 
ance of  respiration  resulted  from  the  piqure.  In  this  case  the  appear- 
ance of  sugar  in  the  urine  was  delayed  and  no  diuresis  was  observed. 

In  all  of  our  experiments  the  procedure  was  as  follows.  The  rabbits 
were  anaesthetized  with  ether.  All  urine  was  so  far  as  possible  pressed 
out  of  the  bladder,  and  tested  for  sugar.  No  sugar  was  found  in  any 
case.  A  sample  of  blood  was  drawn  from  the  femoral  artery  for  the 
gas  analyses.23  Piqure  was  then  performed.  The  animal  was  immedi- 
ately placed  in  the  ventilated  chamber  and  supplied  either  with  air 
alone  or  with  air  and  carbon  dioxide  of  the  percentage  shown  in  the 
table.  The  chamber  was  so  arranged  that  samples  of  blood  could  be 
drawn  and  urine  pressed  out  of  the  bladder  without  removing  the  ani- 
mal's head  and  forequarters  from  the  chamber.  An  attempt  to  obtain 
urine  in  this  way  was  made  every  fifteen  minutes.  The  data  of  ten 
typical  experiments  are  summar'zed  in  the  table  on  page  285. 


IV.  Pancreatic  Diabetes. 

From  the  single  experiment  outlined  below  it  is  apparent  that  when 
glycosuria  is  induced  by  removal  of  the  pancreas  at  first  a  notable 
diminution  of  the  CO2  content  of  the  arterial  blood  occurs.  The  animal 
breathes  excessively  and  later  subnormally.  We  have  no  reason  to 
suppose  that  preventing  the  development  of  acapnia  would  prevent 
glycosuria  or  delay  the  fatal  result  materially.  Even  this  single  ex- 
periment shows,  however,  that  pancreatic  diabetes  does  not  necessarily 
involve  hypercapnia  (cf.  Edie,  Moore,  and  Roaf  on  page  283  of  this 
paper).  The  circulation  fails  rapidly  in  all  such  experiments,  as  Un- 
derhill has  frequently  had  occasion  to  observe,  and  the  venous  blood 
soon  shows  by  its  color  an  almost  entire  lack  of  oxygen.  Thus,  although 
the  arterial  blood  may  be  rich  in  oxygen,  the  tissues  may  be  insuffi- 
ciently supplied  because  of  failure  of  the  circulation.  We  are  not  yet 

23  Barcroft  and  Haldane:  Journal  of  physiology,  1902,  xxviii,  p.  234.  The 
flasks  used  by  us  were  three  times  as  large  as  those  of  Barcroft  and  Haldane,  and 
the  blood  samples  were  3.0  c.c  instead  of  only  1.0  c.c. 


Acapnia  and  Glycosuria.  285 

prepared  to  follow  the  lead  of  those  who  hold  that  deficiency  of  oxygen  is 
not  capable  of  disturbing  sugar  metabolism. 


ARTERIAL  BLOOD  GASES. 


Exp. 
no. 

Before  piqure. 

1  hr.  after 
piqure. 

2  hrs.  after 
piqure. 

7  hrs.  after 
piqure. 

Appear- 
ance of 
glyco- 
suria 
in  min. 

Polyuria. 

o2 

C02 

02 

C02 

c2 

C02 

02 

C02 

1 1 

13.5 

51.1 

13.3 

28.7 

13.5 

45.7 

2  1 

14.5 

58.0 

14.8 

37.9 

14.5 

42.0 

30 

31 

12.0 

40.0 

12.0 

26.3 

8.5 

34.3 

12.8 

30.0 

45 

41 

12.0 

38.9 

16.4 

17,1 

60 

52 

13.0 

36.0 

75 

0 

63 

210 

0 

74 

12.1 

50.0 

14.5 

47.2 

135 

0 

8B 

14.5 

45.8 

7.3 

61.5 

9.2 

69.5 

75 

0 

96 

16.2 

25.7 

15.2 

44.5 

60 

0 

10  7 

1  Breathed  air.    Hypernoea.  2  No  hypernoea. 

3  Breathed  10  per  cent  C02.  4  Breathed  15  per  cent  C02. 

5  Breathed  25  per  cent  C02.  6  Breathed  25  per  cent  C02. 

7  Normal  rabbit  not  anaesthetized;  no  piqure,  breathed.25  per  cent  C02  for  one  hour. 
No  trace  of  sugar  appeared  in  the  urine. 

8  . .  indicates  marked  polyuria,  and  zero  indicates  no  apparent  diuresis. 


Experiment  of  February  14,  igo8.  — 
9.45.    Dog  under  chloroform. 
10.00.    Arterial  gases,  02:19.3;  CO2 42.3  per  cent. 
1 1 . 1 5 .    Pancreas  completely  removed. 
11.30.    Respiration  30  per  minute,  and  full. 
1. 10.    Urine  full  of  sugar. 

1. 15.    Arterial  gases,  O2 119.4  per  cent;  CO2 128.6  per  cent. 
2.00.    Tube  attached  to  trachea  2  cm.  in  diameter  and  130  cm.  long. 
Respiration  very  weak. 

2.55.    0.12  gm.  morphin  sulphate  subcutaneously. 


286 


Yandell  Henderson  and  Frank  P.  Under  hill. 


3.50.    Arterial  gases,  02:23.2  per  cent;  002:31.0  per  cent. 
4.45.    Urine  full  of  sugar.   Respiration  almost  failing. 
5.15.    Saline  saturated  with  CO2  placed  in  abdomen.  Attached 
tube  to  trachea.  Deep  respiration  follows. 
7.30.    Animal  stopped  breathing. 

V.  Piperidin  Diabetes. 

Underhill  has  observed  that  when  piperidin  is  painted  upon  the 
pancreas  a  notable  hyperpncea  followed  ^by  subnormal  breathing 
occurs.  Herter  24  had  previously  demonstrated  that  painting  piperidin 
upon  the  pancreas  results  in  a  marked  glycosuria  which  persists  for 
several  hours.  Although  we  have  performed  but  a  single  experiment, 
it  appears  sufficient  to  show  that  hypercapnia  is  not  a  factor  in  this 
form  of  glycosuria,  but  that,  on  the  contrary,  the  animal  develops  a 
notable  acapnia.  During  the  period  of  subnormal  breathing  which 
follows  the  tissues  may  be  insufficiently  supplied  with  oxygen,  because 
of  the  low  content  of  oxygen  in  the  venous  blood. 

In  the  earlier  experiments  of  Underhill  it  was  found  that  this  form 
of  diabetes  was  prevented  when  oxygen  was  given.  It  is  possible  that 
the  principal  factor  in  this  result  was  the  prevention  of  acapnia,  as 
the  process  of  breathing  into  a  mask  necessarily  involves  more  or  less 
re-breathing.  This  prevention  of  acapnia  would  later  obviate  also 
that  tendency  to  failure  of  respiration  and  consequent  anoxhaemia 
which  were  observed  by  Underhill. 

February  25,  igo8.  —  Dog,  15  kilos. 

11.30.    Chloroform  —  then  ether. 

12.00.  Arterial  gases,  02:19.9;  002:33.7.  Venous  gases,  02:15.2; 
CO2:  40.9. 

12.10.  Urine  free  from  sugar;  blood  pressure,  75  mm.  Hg.  Piperidin 
solution — 10  per  cent  —  painted  on  pancreas.  Less  than  1  c.c.  used. 
Respiration  immediately  accelerated. 

12.15.    Arterial  pressure,  95  mm.;  hyperpncea. 

12.25.    Arterial  pressure,  no  mm.;  coma.   Urine  free  of  sugar. 

1. 10.  Arterial  gases,  02:  18.9  per  cent;  C02:27.o  per  cent.  Venous 
gases,  02:11.1  per  cent;  CO2:  28.0  per  cent. 

1. 1 5.    Arterial  pressure,  130  mm. 

24  Herter:  Medical  news,  1902,  xxx,  p.  865. 


Acapnia  and  Glycosuria. 


287 


1.45.    No  sugar  in  urine.   1  c.c.  piperidin  painted  on  pancreas. 
2.15.    1  c.c.  piperidin  10  per  cent  painted  on  spleen. 
2.45.    Urine  contains  sugar. 

3.20.  Arterial  gases  02:9.9  per  cent;  002:46.9  per  cent.  Venous 
gases,  02:3.8  per  cent;  002:57.2  per  cent. 

3.00  to  3.20.    Very  feeble  respiration.   Animal  barely  alive. 

3.25.  Slow  heart  beat;  65  per  minute;  respiration  poor.  Arterial 
pressure  varying  from  50  to  120  mm.   Urine  contains  sugar. 

3.50.    Animal  died  of  failure  of  respiration. 

VI.  Glycosuria  after  Laparotomy  and  after  Excessive 
Artificial  Respiration. 

It  has  been  shown  by  Henderson  25  that  when  the  intestines  are 
handled  in  a  current  of  warm  moist  air  acapnia  develops.  The  first 
of  the  two  experiments  given  below  indicates  that  hyperglycemia  and 
glycosuria  may  follow  the  acapnia. 

The  most  direct  procedure  for  the  experimental  production  of  acap- 
nia is  excessive  artificial  respiration.  Henderson 26  has  found  that 
artificial  respiration  administered  with  a  hand  bellows  while  the  thorax 
is  intact,  usually  produces  little  or  no  acapnia.  The  elastic  recoil  of 
the  thorax  in  expiration  under  these  conditions  is  so  slow  that  an  exces- 
sive ventilation  is  difficult  to  obtain.  It  is  necessary  that  the  apparatus 
employed  for  a  dog  with  intact  thorax  should  not  only  inject  fresh  air, 
but  also  that  it  should  quickly  and  forcibly  suck  out  again  the  other- 
wise slowly  expired  air.  Excessive  ventilation  can,  howTever,  be  ob- 
tained merely  with  a  hand  bellows  after  the  thorax  has  been  opened. 
Under  these  conditions  the  lungs  collapse  and  expel  their  air  rapidly 
in  the  intervals  between  the  strokes  of  the  bellows.  This  method  of 
inducing  acapnia  was  employed  in  the  second  experiment  reproduced 
below.  No  urine  was  obtained  after  acapnia  had  developed,  but  the 
blood  sugar  exhibited  a  notable  increase. 

Aeration  of  Intestine. 

November  ig,  igo8.  —  Vigorous  bulldog;  weight,  7.5  kilos. 
10.30.    Etherized  until  10.45. 

25  Henderson,  Y.:  This  journal,  1909,  xxiv,  p.  66. 

26  Henderson,  V.:  This  journal,  1910,  xxv,  p.  322. 


288 


Yandell  Henderson  and  Frank  P.  Underbill. 


n.oo.  Arterial  gases,  02:23.3  per  cent;  C02:3Q.6  per  cent.  Blood 
sugar,  0.18  per  cent;  respiration,  42.  Pulse,  160;  urine  free  from 
sugar.    NH4— N  fraction  :  5.0  per  cent. 

11. 10.  Tracheotomized;  abdomen  opened;  intestines  aerated; 
shallow  respiration.    Very  little  ether  necessary. 

12.25.  Gases  in  blood  from  mesenteric  vein;  oxygen,  10.3  per  cent; 
002:31.3  per  cent. 

12.28.    Comatose;  arterial  gases,  02:17.5  per  cent;  C02:25.8  per  cent. 

I.  00.  Respiration,  18;  pulse,  170.  Good  arterial  pressure;  urine 
full  of  sugar;  blood  sugar,  0.30  per  cent;  NH4— N  fraction  of  urine,  8.3 
per  cent. 

December  3,  1907.  —  Young  dog,  10  kilos.   First  chloroform;  then  ether; 
resists  anaesthesia. 

10.50.  Arterial,  02:17.0  per  cent;  002:36.1  percent.  Venous,  02: 
14.1  per  cent;  002:38.4  per  cent.  Arterial  pressure,  150  mm.;  pulse, 
150.    Rapid  respiration. 

II.  15.  Thorax  opened;  rapid  and  full  artificial  respiration  ad- 
ministered. 

11.30.    Urine  free  from  sugar. 

12.30.    No  anaesthetic  necessary;  animal  in  profound  shock.  Arte- 
rial gases,  O2: 17.2;  CO2: 14.5  per  cent.  Venous,  O2:  6.9  per  cent;  CO2: 
33.9  per  cent.   Intestines  relaxed;  not  irritable;  bladder  empty. 
^.       10.50.    Blood  sugar,  0.16  per  cent. 
'  (12.30.    Blood  sugar,  0.22  per  cent. 

VII.  Conclusions. 

Acapnia  is  a  frequent  concomitant  of  glycosuria  or  at  least  of  hyper- 
glycaemia  both  under  clinical  and  experimental  conditions.  In  some 
artificial  forms  of  diabetes  prevention  of  acapnia  obviates  disturbance 
of  the  sugar-regulating  function. 

We  believe  that  glycosuria  after  etherization  is  due  to  acapnia,  and 
that  traumatic  and  emotional  glycosurias  also  are  usually  due  to  this 
cause. 

The  work  of  previous  investigators  is  here  quoted  to  show  (a)  that  in 

diabetic  coma  an  acute  acapnia  occurs;  (b)  that  this  is  a  true  acapnia 

resulting  from  hyperpncea  and  is  not  merely  due  to  the  expulsion  of 

CO2  from  the  bicarbonates  of  the  blood  by  acids;  (c)  that  in  acidosis 
+ 

the  acidity,  i.  e.  (H),  of  the  blood  is  probably  below  normal,  instead  of 


Acapnia  and  Glycosuria. 


289 


above,  as  usually  assumed;  (d)  that  the  hyperpncea  of  diabetic  coma  is 
induced  by  the  ethereal,  not  the  acid,  acidosis  bodies,  e.  g.,  acetone. 

In  conclusion  we  would  point  out  that  it  is  often  impossible  to  infer 
the  interior  conditions  of  tissue  respiration  from  the  external  condi- 
tions to  which  an  animal  may  be  exposed.  The  only  certain  cri- 
terion of  acapnia  or  hypercapnia  is  analysis  of  alveolar  air  or  blood 
gases.  The  only  certain  criterion  of  insufficient  oxygen  supply  to  the 
tissues  is  the  demonstration  that  the  venous  blood  contains  no  oxygen 
or  only  a  minimal  amount. 


TRANSACTIONS  OF  THE 

CONNEGTIGUT  ACADEMY  OF  ARTS  AND  SGIENGES 


Incorporated  A.D.  1799 


VOLUME  I  6,  PAGES  247-382  APRIL,  1911 

Nutrition  Investigations 

on  the 

Carbohydrates 

I  of 

Lichens,  Algae,  and  Related 
Substances 


BY 

MARY  DAVIES  SWARTZ 

FROM  THE  LABORATORY  OF  PHYSIOLOGICAL  CHEMISTRY 
SHEFFIELD  SCIENTIFIC  SCHOOL 

YALE  UNIVERSITY 

NEW  HAVEN,  CONNECTICUT,  U.  S.  A. 


YALE  UNIVERSITY  PRESS 
NEW  HAVEN,  CONN. 
1911 


COMPOSED  AND  PRINTED  AT  THE 

WAVERLY  PRESS 
The  Williams  &  Wilkins  Company 
Baltimore,  U.  S.  A. 


CONTENTS. 
I.  INTRODUCTION. 

PAGE 

Lichens,  Algae,  Tree  Bark  and  Certain  Tubers  and  Foodstuffs   253 

II.    HISTORICAL  PART. 

Introduction   259 

Cellulose    262 

(a)  Occurrence  and  Nature   262 

(b)  Occurrence  of  Cytases  (Cellulases)   264 

(1)  In  the  Vegetable  Kingdom   264 

(2)  In  Lower  Animals   266 

(3)  In  Higher  Animals  ,   267 

(c)  Digestion  and  Utilization   268 

(1)  By  Animals   268 

(2)  By  Man   269 

The  Pentosans   272 

(a)  Occurrence  and  Nature   272 

(b)  Role  in  Plant  Physiology   274 

(c)  Occurrence  of  Pentosanases   275 

(1)  In  the  Vegetable  Kingdom   275 

(2)  In  Lower  Animals   276 

(3)  In  Higher  Animals   276 

(d)  Digestion  and  Utilization   278 

(1)  By  Animals   278 

(2)  By  Man   278 

The  Galactans   282 

(a)  Occurrence  and  Nature   282 

(b)  Occurrence  of  Galactanases   284 

(1)  In  the  Vegetable  Kingdom   284 

(2)  In  the  Animal  Kingdom   285 

(c)  Digestion  and  Utilization  by  Animals  and  Man   285 

The  Mannans   289 

(a)  Occurrence  and  Nature   289 

(b)  Occurrence  of  Mannanases   291 

(1)  In  the  Vegetable  Kingdom   291 

(2)  In  the  Animal  Kingdom   292 

(c)  Digestion  and  Utilization  by  Animals  and  Man   293 

The  Levulans   295 

(a)  Occurrence  and  Nature   295 

(b)  Occurrence  of  Levulanases   296 

(1)  In  the  Vegetable  Kingdom   296 

(2)  In  the  Animal  Kingdom   297 

(c)  Digestion  and  Utilization  by  Animals   297 

249 


250  Contents 

The  Dextrans   300 

(a)  Occurrence  and  Nature   300 

(b)  Occurrence  of  Dextranases   301 

(1)  In  the  Vegetable  Kingdom   301 

(2)  In  the  Animal  Kingdom   302 

(c)  Digestion  and  Utilization  by  Animals  and  Man   302 

III.  EXPERIMENTAL  PART. 

Introduction   306 

Chemical  Investigations. 

General  Methods   307 

Pentosan  Preparations   309 

(a)  Dulse  (Rhodymenia  palmata)   309 

(b)  Hawaiian  Seaweeds   313 

(1)  Limu  Lipoa  (Haliseris  pardalis)   313 

(2)  Limu  Eleele  (Enteromorpha  intestinalis)  313 

(3)  Limu  Pahapaha  {Ulza  lactuca,  etc)   314 

Galactan  Preparations   314 

(a)  Irish  Moss  (Chrondus  crispus)   314 

(b)  Hawaiian    Seaweeds   316 

(1)  Limu  Manauea  (Gracilaria  coronopifolia)   316 

(2)  Limu  Huna  (Hypnea  nidijica)   316 

(3)  Limu  Akiaki  (Ahnfeldtia  concinna)   316 

(4)  Limu  Uaualoli  (Gymnogongrus  vermicularis  Americana, 
etc)   316 

(5)  Limu  Kohu  (Asparagopsis  sanfordiana)   316 

(c)  Slippery  Elm  (Ulmus  fulva)   317 

A  Mannan  Preparation — Salep  (Orchis)   318 

A  Levulan  Preparation — Sinistrin  (from  Scilla  Maritima)   321 

Summary    322 

BACTERIOLOGICAL  INVESTIGATIONS. 

Introduction    323 

Trials  with  pure  cultures  of  aerobes   324 

Trials- with  mixtures  of  aerobes   325 

Trials  with  anaerobes   327 

Discussion  and  summary   328 

PHYSIOLOGICAL  INVESTIGATIONS. 

Introduction    331 

Experiments  with  Enzymes   332 

Parental  Injections   332 

(a)  Methods  and  Technique   332 


Contents  251 

(b)  Subcutaneous  and  Intraperitoneal  Injections   335 

(1)  Dulse   335 

(2)  Irish  Moss   336 

(3)  Salep   338 

(4)  Sinistrin   340 

Feeding  Experiments   342 

(a)  Methods  and  Technique   342 

(b)  Digestibility  of  Pentosans   344 

(1)  Dulse   345 

(2)  Limu  Eleele   346 

(3)  Limu  Pahapaha   347 

(4)  LimuLipoa   347 

(c)  Digestability  of  Galactans   348 

(1)  Irish  Moss   349 

(2)  Limu  Ma'nauea   350 

(3)  Limu  Huna   351 

(4)  Limu  Akiaki   351 

(d)  Digestibility  of  Mannan  1 ...  353 

(1)  Salep   354 

Discussion  and  Summary   356 

IV.  CONCLUSIONS. 

V.  BIBLIOGRAPHY. 

Lichens  and  Algae — Composition  and  uses   365 

Cellulose   366 

Pentosans   369 

Galactans   373 

Mannans   376 

Levulans   379 

Dextrans    381 


This  paper  has  been  prepared  from  the  author's  dissertation  submitted  for 
the  degree  of  doctor  of  philosophy,  Yale  University,  1909. 


I.  INTRODUCTION. 


Lichens,  Algae,  Tree  Bark  and  Certain  Tubers  as  Foodstuffs. 

From  the  earliest  times,  the  food  of  man  has  included  lichens  and 
algae,  and  even  the  tender  branches  and  inner  bark  of  certain  trees 
and  shrubs,  such  as  elm,  birch,  pine,  and  the  staff-tree  or  bitter-sweet 
{Celastrus  scandens).  When  the  bark  of  trees  is  so  used,  it  is  freed 
from  cork  and  the  hard  outer  rind;  is  cleaned,  dried,  mixed  with  more 
or  less  meal,  and  made  into  "bark  bread."  Such  substitutes  for 
bread  are  commonly  resorted  to  only  in  northern  lands  where  there  is 
scarcity  of  cereal  crops,  or  in  other  regions  during  periods  of  famine. 
Johnson  (7)  records  that  elm  bark  is  so  employed  in  some  continental 
countries,  and  Dillingham  (4)  relates  that  certain  tribes  of  North 
American  Indians,  'in  times  of  extreme  dearth,  were  accustomed  to 
keep  body  and  soul  together  by  boiling  and  eating  the  bark  of  the 
staff-tree.'  Poulsson  (17)  states  that  in  Finland  and  northern  Russia, 
sphagnum  mosses  are  similarly  employed;  and  Schneider  (21)  agrees 
with  these  other  writers,  saying  that  in  general  lichens  are  used  as 
articles  of  diet  only  in  cases  of  special  need,  principally  because  all 
lichens  contain  a  bitter  principle,  which  not  only  gives  an  unpleasant 
flavor  and  is  difficult  to  remove,  but  also  exerts  an  irritating  effect 
upon  the  digestive  tract,  causing  inflammation.  Nevertheless,  in  the 
northern  parts  of  the  Scandinavian  Peninsula,  where  cereal  crops  are 
always  scanty  or  uncertain,  great  interest  attaches  to  two  species  of 
lichen  widely  distributed  through  Europe,  and  through  Arctic  and 
Antarctic  regions:  namely,  Celraria  islandica  and  Cetraria  nivalis, 
which,  as  Poulsson  (17)  observes,  'have  been  considered  nutritive 
and  easily  digestible  since  olden  times. '  Cetraria  islandica,  whitened 
and  freed  from  its  bitter  principle  by  washing  with  dilute  alkali,  is  a 
rather  appetizing  substance;  it  has  sometimes  been  used  as  a  foodstuff 
by  Polar  navigators,  and  Dr.  Hansteen,  chief  lecturer  in  the  Agricul- 
tural school  at  Aas,  Norway,  has  gone  so  far  as  to  prophesy  that  moss 
is  destined  to  become  the  great  popular  food  for  the  masses,  because 
of  its  cheapness  and  nutritive  properties. 

Of  marine  algae,  many  tons  are  gathered  and  eaten  annually  in 
various  parts  of  the  world,  the  largest  quantities  being  consumed 

253 


254 


Mary  Davies  Swartz, 


by  the  Japanese,  Chinese,  and  Hawaiians.  These  algae  are  found 
in  great  variety  and  widely  distributed.  In  Japan,  the  general  name 
applied  to  them  is  "Nori,"  which  is  also  given  to  several  prepared 
products.  According  to  H.  M.  Smith  (23),  the  most  important  Japan- 
ese seaweed  preparations  are:  "Kanten,"  or  seaweed  isinglass,  made 
from  various  species  of  Gelidium,  the  principal  one  being  Gelidium 
comeum,  often  adulterated  with  similar  seaweeds;  "Kombu"  made 
from  Kelps,  especially  numerous  species  of  Laminaria,  Arthothamnus , 
and  Alaria;  "Amanori,"  from  species  of  Porphyra;  and  "Wakame," 
from  Undaria  pinnatifida. 

Kanten  is  used  largely  for  food,  in  the  form  of  jellies,  and  as  an  adju- 
vant of  soups  and  sauces.  According  to  H.  M.  Smith  (23),  it  is  also 
employed  in  foreign  countries  'in  jellies,  candies,  pastries,  and  many 
desserts,  in  all  of  which  it  is  superior  to  animal  isinglass.'  It  has 
recently  also  attained  popularity  as  a  therapeutic  agent  in  chronic 
constipation,  being  sold  under  various  trade  names,  either  plain  or 
impregnated  with  laxative  drugs,  as  cascara  or  phenolphthalein.1 
Kombu  enters  into  the  dietary  of  every  Japanese  family,  being  cooked 
with  meat,  soups,  etc.,  and  also  served  as  a  vegetable,  or  made  into  a 
relish  with  Soy-bean  sauce.  Amanori  is  eaten  fresh  or  else  is  chopped 
and  sun-dried  in  thin  sheets,  which  are  toastsd  over  a  fire  before 
eating.  The  crisp  amanori  is  crushed  between  the  hands  and  dropped 
into  sauces  or  soups  to  impart  flavor;  or  broken  into  pieces,  dipped  in 
sauce  and  eaten  alone.  Sheets  of  amanori,  spread  with  boiled  rice 
and  covered  with  strips  of  meat  or  fish,  are  rolled  and  cut  into  trans- 
verse slices,  and  take  the  place  of  the  American  sandwich.  Wakame 
is  eaten  as  a  salad,  or  cooked  like  amanori. 

In  Hawaii,  edible  algae  are  called  "limu. "  Of  these  there  are  over 
seventy  distinct  species  used  for  food,  more  than  forty  being  in  general 
use  (18).  Tons  of  limu  are  gathered  for  eating  in  Hawaii  annually, 
and  large  quantities  are  also  imported  from  the  Orient  and  San  Fran- 
cisco. Some  idea  of  the  extent  of  their  use  may  be  gained  from  the 
following  statement  by  Miss  Reed  (18):  "  Ancient  Hawaiians  prob- 
ably seldom  ate  a  meal  without  some  kind  of  limu,  and  even  today  no 
Hawaiian  feast  is  considered  quite  complete  without  several  varieties 
served  as  a  relish  with  meats  or  poi."2  Since,  with  the  exception  of  a 
few  experiments  reported  by  Oshima  (15)  and  Saiki  (20),  there  are  no 


lCf.  Galactans,  p.  283. 

2Poi  is  a  thick  paste  made  from  the  root  of  the  taro  plant,  and  takes  the  place  of 
rice  or  bread  in  the  native  diet. 


Nutrition  1  nvestigations. 


data  upon  the  digestibility  of  marine  algae,  an  investigation  of  some 
of  these  Hawaiian  limu  seemed  highly  desirable;  and  through  the 
kindness  of  Miss  Reed,  a  number  have  been  obtained  for  this  purpose. 
Their  occurrence  and  uses  will  therefore  be  described  in  some  detail.1 
These  limu  are  washed  carefully  after  gathering,  salted,  and  usu- 
ally broken,  pounded,  or  chopped  into  small  pieces.  They  may  then 
be  eaten  uncooked,  as  a  relish  with  poi,  meats  or  fish;  boiled  with  meats; 
put  into  soups  for  thickening  or  flavoring;  or  roasted  with  pig  in  a  pit. 
Served  raw  and  crisp,  they  take  much  the  same  place  in  the  diet  as 
our  salads.  Among  the  most  popular  varieties  are  Limu  Eleele  (Enter  o- 
morpha  of  various  species),  Limu  Kohu  (Asparagopsis  sanfordiana) 
and  Limu  Lipoa  (Haliseris  partialis) .  Next  in  favor  come  Limu  Ma- 
nama (Gracilaria  cor onopif olio),  Limu  Huna  (Hypnea  nidifica)  and 
Limu  Akiaki  (Ahnfeldtia  concinna).  Limu  Pahapaha  (Ulva  fasciata 
and  Ulva  lactuca)  is  widely  distributed  but  not  very  popular.  Limu 
Uaualoli  (Gymnogongrus  vermicularis  americana  and  Gymnogongrus 
disciplinalis)  is  limited  to  certain  islands,  and  hence  not  in  such  gen- 
eral use  and  favor  as  some  of  the  others. 

Limu  eleele  is  a  great  favorite,  forming  a  part  of  every  native 
feast.  It  is  generally  eaten  uncooked,  sometimes  being  dropped  into 
hot  gravy,  broth  or  meat  stews  just  before  serving.  Limu  kohu  is 
always  pounded  in  cleaning  to  free  it  from  bits  of  coral  and  soaked  24 
hours  in  fresh  water  to  remove  the  bitter  iodine  flavor.  It  becomes 
slightly  fermented  and  acquires  a  somewhat  sour  taste.  Limu  lipoa 
is  popular  on  account  of  its  penetrating  spicy  flavor,  and  is  frequently 
used  as  a  condiment,  taking  the  place  of  sage  and  pepper  in  Hawaiian 
foods.  Limu  huna  is  especially  prized  for  boiling  with  squid  or  octo- 
pus, though  limu  manauea  and  limu  akiaki  are  often  used  as  substi- 
tutes. These  limus,  as  well  as  limu  kohu,  yield  large  amounts  of 
mucilaginous  extract  on  boiling,  limu  manauea  being  considered  es- 
pecially fine  for  thickening  chicken  broth. 

Many  of  the  seaweeds  used  in  Hawaii  and  Japan  occur  also  along 
the  coasts  of  the  United  States  and  Europe,  and  are  to  some  extent 
used  as  food  in  both  regions.  The  very  species  of  Gelidium  from 
which  the  Japanese  prepare  their  Kanten  grow  in  abundance  on  our 
Pacific  coast.  Irish  moss  (Chondrus  crispus),  the  "Tsunomata"  of 
Japan,  has  long  had  considerable  commercial  value  as  a  foodstuff  in 
Ireland.  In  this  country  it  is  found  from  North  Carolina  to  Maine, 
being  especially  abundant  north  of  Cape  Cod.    After  cleansing,  cur- 


sor fuller  description  see  Reed  (18). 


256 


Mary  Dames  Swartz, 


ing,  and  bleaching  it  is  to  some  extent  used  for  making  blanc  mange 
or  a  demulcent  for  coughs.  Through  the  kindness  of  Dr.  C.  F.  Lang- 
worthy,  Nutrition  Expert,  United  States  Department  of  Agriculture, 
I  have  obtained  the  following  interesting  data  concerning  the  use  of 
Irish  moss,  from  the  Journal  of  the  South-Eastern  Agricultural  Col- 
lege, Wye,  Kent  (1):  "  Professor  D.  Houston,  of  the  Royal  College  of 
Science,  Dublin,  has  favored  us  with  the  following  notes  on  this  sub- 
ject: 

Chondrus  crispus  (carrageen,  or  Irish  moss)  is  a  seaweed  plentifully  distributed 
along  our  northern,  western  and  southern  coasts.  It  is  gathered  and  sold  to  local 
chemists,  who  retail  it,  in  some  parts  at  all  events,  at  6d.  per  pound.  It  is  used  by 
many  people  as  an  article  of  food  in  the  west,  and  generally  for  colds,  for  which  pur- 
pose it  is  boiled  in  milk. 

Several  of  my  students  tell  me  that  it  is  used  for  feeding  weak  calves  and  with 
striking  results,  bringing  about  an  alteration  of  condition  within  four  days.  One 
student  tells  me  that  in  one  case  at  his  own  farm  a  batch  of  twelve  calves  took  a 
kind  of  wasting  disease,  and  nine  died;  the  other  three  on  the  verge  of  death  were 
given  this  plant,  and  all  three  recovered.  It  is  prepared  by  putting  one  pound  of 
the  "weed"  in  a  net  bag  and  boiling  in  a  gallon  of  water.  The  water  on  cooling 
sets  to  a  jelly.  The  calves  are  given  one  glass  of  jelly  in  their  milk  each  meal 
and  wonderful  results  are  said  to  be  obtained." 

The  high  proportion  of  mineral  matter  is  noteworthy;1  but  without 
making  a  fuller  investigation,  it  is  impossible  to  say  precisely  wherein 
lies  the  value  of  this  seaweed. 

Purple  laver  (Porphyra  laciniata),  a  source  of  Japanese  amanori, 
is  found  in  abundance  on  the  rocky  shores  of  America  and  Europe 
generally ;  but  it  is  not  used  in  this  country  save  sparingly  by  the  Chi- 
nese, who  usually  import  it  directly  from  China,  and  by  some  of  the 
Indians  of  our  northwest  coast.  In  Ireland  it  is  known  as  'sloak,' 
and  is  boiled  and  served  with  butter,  pepper,  and  vinegar  as  an  ac- 
companiment of  cold  meats,  or  is  served  with  leeks  and  onions. 

Dulse  (Rhodymenia  palmata)  is  found  abundantly  on  rocky  shores 
both  in  this  country  and  in  Ireland.  It  is  very  abundant  in  New 
England,  where  it  is  rough-dried  in  the  sun  and  eaten  as  a  relish.  In 
Philadelphia  it  is  called  sea-kale  and  eaten  as  a  vegetable.  In  Scot- 
land it  has  long  been  used  both  in  the  fresh  state  and  dried.  In  the 
Scotch  Highlands,  "a  dish  of  dulse  boiled  in  milk  is,"  it  is  said,  "the 
best  of  all  vegetables."  In  Ireland,  it  is  eaten  with  fish  or  boiled  in 
milk  with  rye  flour.  Purple  dulse  (Iridea  edulis),  which  occurs  on 
the  Pacific  coast,  is  often  eaten  like  Rhodymenia  palmata. 


lCf.  Analysis  of  Chondrus  crispus,  p.  254. 


Nutrition  Investigations. 


257 


Besides  such  lichens  and  algae,  and  the  bark  of  trees,  various  tubers 
are  used  as  food  for  man.  In  Japan,  the  tubers  of  Hydrosme  rivieri 
(Conophallus  Konjaku)  are  extracted  with  lime  water,  and  the  result- 
ing gelatinous  mass  is  cut  into  small  cakes.  These,  cooked  with 
"shoyu"  or  Soy-bean  sauce  form  a  common  article  of  diet.  The 
tubers  of  many  species  of  Orchis  and  Eulophia,  native  to  Turkey,  the 
Caucasus,  Asia  Minor  and  the  greater  part  of  Central  and  Southern 
Europe,  furnish  a  food  material  known  as  Salep.  The  small  ovoid, 
oblong  or  palmate  tubers  are  decorticated,  washed,  heated  till  horny 
and  semi-transparent,  and  finally  dried.  An  abundant  mucilaginous 
extract  is  obtained  by  macerating  the  bulbs  in  water.  Frequently 
the  tubers  are  ground  to  powder,  and  the  powder  used  like  sago  or 
tapioca.  Royal  salep,  said  to  be  used  as  food  in  Afghanistan,  is  pre- 
pared from  Allium  Macleanii.  A  former  instructor  in  the  American 
College  for  Girls,  in  Constantinople,  reports  that  salep  is  a  very  com- 
mon article  of  diet  in  Turkey.  It  is  sold  in  the  markets  in  powdered 
form,  and  is  made  into  a  sort  of  sweetened  gruel  with  milk.  Not  only 
is  it  used  as  a  warm  drink  in  the  household,  much  as  we  use  cocoa  or 
chocolate,  but  it  is  also  sold  in  the  streets  by  venders,  who  either 
stand  in  booths  along  the  way,  or  go  about  carrying  huge  brass  urns 
strapped  to  their  shoulders,  clinking  their  cups  and  calling  "  Taze- 
Sahlep!"1  It  is  especially  popular  in  districts  of  the  city  where  peo- 
ple work  late  at  night.  In  the  month  of  Ramazon,  the  time  of  all-day 
fasting,  hot  salep  finds  a  ready  sale  at  night.  It  is  no  uncommon 
thing  to  see  the  workman  standing  with  his  salep  cup  in  hand,  waiting 
for  the  firing  of  the  sunset  cannon. 

In  spite  of  the  fact  that  there  have  been  almost  no  scientific  inves- 
tigations as  to  the  digestibility  of  such  mucilaginous  plant  substances 
there  seems  to  be  a  special  virtue  attached  to  mucilages  in  the  popular 
mind.  The  prevailing  impression  is  shown  in  some  of  the  following 
remarkable  statements.  The  United  States  Dispensatory,  1908,  not 
only  says  that  the  mucilaginous  extract  of  slippery  elm  bark  (Ulmus 
fulva,  Michaux)  is  nutritious,  but  adds,  "  We  are  told  that  it  has  proved 
sufficient  for  the  support  of  life  in  the  absence  of  other  food."  Of 
salep  Smith  (25)  says  in  his  dictionary  of  economic  plants:  "It  con- 
tains a  chemical  substance  called  bassorin,  which  is  said  to  contain 
more  nutritious  matter  than  any  other  vegetable  product,  one  ounce 
per  diem  being  sufficient  to  sustain  a  man"!  The  United  States  Dis- 
pensatory also  assures  us  that  salep  is  "highly  nutritious."  Johnson 


^resh  salep. 


258 


Mary  Davies  Swartz. 


(7)  particularly  recommends  Iceland  moss  (Cetraria  islandica)  as  a 
diet  for  consumptives,  as  "it  seems  to  be  both  extremely  nutritious 
and  very  easy  of  digestion,  though  of  course,  only  capable  of  use  as  a 
substitute  for  starchy  matters."  In  regard  to  Irish  moss  (Chondrus 
crispus),  he  is  a  little  more  uncertain.  "It  is  much  used  for  invalids, 
especially  in  cases  of  consumption,  but  with  doubtful  advantage  when 
substituted  for  more  nutritious  food."  Schneider  (21)  says  of  Ice- 
land moss:  "Inhabitants  of  Iceland,  Norway,  and  Sweden  mixed  this 
lichen  with  various  cereals  and  mashed  potatoes,  from  which  an  un- 
commonly healthful  bread  was  prepared."  Until  the  matter  has  been 
thoroughly  investigated,  we  must  suspend  our  judgment  as  to  the  ac- 
curacy of  such  statements.  After  a  few  metabolism  experiments, 
Oshima  (15)  far  more  conservatively  remarks  concerning  the  algae  of 
Japan:  "Their  actual  value  doubtless  depends  in  considerable  measure 
upon  the  mineral  salts  they  contain." 

In  view  of  the  scarcity  of  any  scientific  investigations  as  to  the  be- 
havior of  all  these  substances  in  the  body,  further  experiments  upon 
their  nature  and  digestibility  seem  highly  desirable,  since  they  are  not 
only  widely  distributed,  and  already  form  a  considerable  portion  of 
the  diet  of  many  persons;  but  because,  if  they  possess  any  real  nutri- 
tive value,  a  wider  use  of  such  comparatively  cheap  materials  would 
be  an  economic  advantage;  and  because,  under  the  prevailing  notions 
as  to  their  food  value,  they  are  sometimes  relied  upon  as  a  source  of 
nutriment  in  diseases  (as  diabetes)  where  the  character  of  the  diet  is 
particularly  important.  The  present  work  has  been  undertaken  to 
throw  some  light  on  this  interesting  subject.  A  survey  of  the  litera- 
ture shows  that  even  the  chemical  nature  of  many  of  these  algae  has 
scarcely  been  investigated;  and  if  this  were  known,  we  should  still  be 
under  the  necessity  of  studying  their  behavior  in  the  animal  body, 
for  it  is  impossible  to  tell  from  chemical  analysis  alone  whether  a 
given  substance  will  or  will  not  prove  digestible,  as  Rubner  has  long 
since  warned  us. 


II. 


HISTORICAL  PART. 


Introduction. 

According  to  the  current  practice  of  agricultural  analysts,  the  car- 
bohydrates of  plants  are  reported  as  crude  fiber  and  nitrogen-free 
extract.  Crude  fiber  is  the  term  applied  to  the  resistant  mixture  form- 
ing the  mature  cell  wall,  shown  as  long  ago  as  1864  by  Henneberg  and 
Stohman  (41)  to  have  no  definite  chemical  composition.  It  is  there- 
fore not  identical  with  cellulose,  but  consists  of  a  mixture  of  cellulose 
with  incrusting  substances,  lignin  and  cutin,  the  relative  proportions 
of  which  have  recently  been  exhaustively  studied  by  Konig  (51), 
Fiirstenberg  (39),  and  Murdfield  (63).  Cellulose  is  the  chief  consti- 
tuent; the  other  two  are  usually  present  in  varying  proportions. 

Schulze  (74)  to  whom  much  of  our  knowledge  of  the  composition  of 
the  plant  cell  wall  is  due,  has  classified  the  carbohydrates  of  the  nitro- 
gen-free extract  as  follows: 

Water-soluble  carbohydrates.    To  this  class  belong  the  mono-, 

di-,  and  tri-saccharides,  and  some  soluble  polysaccharides. 
Carbohydrates  insoluble  in  water,  but  yielding  sugar  under 
the  action  of  diastase.    The  chief  member  of  this  group  is 
starch. 

Carbohydrates  insoluble  in  water  and  resistant  to  the  action 
of  diastase,  never  being  changed  by  it  into  sugar.  This 
group  is  called  the  Hemicelluloses. 

The  term  hemicellulose,  as  used  by  recent  writers1  seems  to  be  inter- 
preted to  include  some  polysaccharides  of  the  first  group.  It  is  there- 
fore used  here  as  a  group  name  for  those  carbohydrates  which  are  dis- 
tinguished from  cellulose  by  being  capable  of  hydrolysis  on  boiling 
with  dilute  mineral  acids,  and  from  the  other  polysaccharide  carbohy- 
drates by  not  being  readily  digested  by  diastase.  According  to  the 
kind  of  sugar  yielded  on  hydrolysis,  the  hemicelluloses  are  designated 
as  Pentosans  or  Hexosans,  the  latter  including  Galactans,  Mannans, 
Dextrans,  Levulans,  etc.    After  a  general  review  of  the  chemical 


ii. 


III. 


^.g.,  Lohrisch. 


259 


260  Mary  Davies  Swartz, 

nature  of  lichens  and  algae,  each  of  these  classes  will  be  discussed 
separately  in  detail. 

The  percentage  composition  of  some  common  species  of  algae  is 
shown  in  the  following  table: 


FOOD  MATERIAL. 

WATER. 

PROTEIN. 

FAT. 

CARBOHYDRATES. 

ASH. 

Nitrogen- 
free 
Extract. 

Crude 

I.* 

Cystophyllum  fusiform, 

15 

74 

11 .37 

.49 

54.84 

17.56 

Ecklonia  bicyclis,  dried. . 

18 

75 

9.58 

.46 

51.63 

9.79 

9.79 

Enteromorpha  linza, 

dried  (Limu  eleele)... 

13 

53 

19.35 

1 .73 

46 

.18 

19.21 

Laminaria  sp.,  dried..  .  . 

23 

08 

7. 11 

.87 

47 

.70 

21.24 

Porphyra  laciniata, 

13 

98 

33.75 

1.30 

41 

.22 

t9.75 

Ulopteryx  pinnatinda, 

dried  

18 

92 

11.61 

.31 

37.81 

31.35 

H.t 

Ahnfeldtia  concinna, 

fresh  (Limu  akiaki)..  . 

80 

00 

1.4 

0.0 

14.4 

4.2 

Ulva  fasciata  and  U. 

Jactuca,  fresh  (Limu 

pahapaha)  

80 

00 

3.7 

0.0 

12.5 

3.8 

Gracilaria  coronopifolia, 

fresh  (Limu  manauea) 

80 

00 

1.8 

0.0 

14 

.1 

4.1 

IILJ 

Chondrus  Crispus,  dried. 

13 

40 

13.06 

2.59 

54.16 

2.57 

14.22 

IV.  § 

0.32 

1.2 

43.3 

5.3 

2.2 

*Oshima  (15). 

t  Reed  (18),  (calculated  on  uniform  water  basis). 
tAnnet  (1). 

§  Schmidt  (24),  first  studied  the  ash  and  reported  a  notable  amount  of  calcium  and  potassium 
phosphates.    He  found  no  nitrogen.    Blondeau  (3)  reported  21.36  per  cent  nitrogen. 
||  Brown  (334). 

Until  1905  the  chemical  nature  of  the  constituents  of  algae  had 
received  little  attention.  Analyses  of  many  species  of  algae  from 
Japan  and  China  were  reported  recently  by  Konig  and  Bethels  (8), 
the  results  of  which  are  given  in  the  following  table  on  page  255. 

According  to  Oshima  and  Tollens  (16)  the  carbohydrates  of  Por- 
phyra laciniata  consist  largely  of  anhydrides  of  d-mannose  and  i-ga- 
lactose.  Miither  and  Tollens  (13)  studying  various  species  of  Fucus 
(F.  vesiculosus,  F.  nododus,  F.  serratus),  Laminaria,  and  Chondrus 
crispus,  found  a  methyl-pentosan  (fucosan),  in  Fucus  and  Laminaria; 
and  glucose,  fructose,  galactose  and  pentose  groups  in  Chondrus 
Krefting  reports  a  reserve  carbohydrate  in  Laminaria  digitata  in  win- 


Nutrition  Investigations . 


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262 


Mary  Dames  Swartz, 


ter  only,  which  yields  d-glucose.  The  algae  investigated  are  thus  all 
seen  to  yield  pentoses,  very  frequently  fructose  and  methyl-pentose, 
sometimes  glucose  and  galactose. 

Lichens  are  symbiotic  forms  embracing  algae  and  fungi.  Because 
of  this  symbiotic  nature,  they  exhibit  great  variety  in  composition. 
From  the  investigations  of  Escombe  (6),  Ulander  and  Tollens  (27), 
Karl  Muller  (11),  Nilson  (14),  Wisselingh  (29)  and  others,1  it  appears 
that  the  cell  walls  are  usually  of  cellulose,  but  occasionally  of  chitin.2 
Many  species  yield  on  extraction  with  hot  water  a  gelatinizing  sub- 
stance, which  Berzelius  (2)  in  1808  named  "  Flechtenstarke  "  (lichenin), 
but  which  later  investigators3  have  shown  to  be,  not  a  single  substance, 
but  a  number  of  related  carbohydrates  yielding  dextrose,  such  as 
lichenin  from  Cetraria  and  Ramalina  fraxinea,  and  evernin  from  Ever- 
nia  prunastre,  usnin  from  Usnea  barbata.  Other  species,  on  the  con- 
trary yield  little  dextran,  but  mannan,  galactan,  pentosan  and  methyl- 
pen  tosan  in  varying  proportions.  The  table  on  page  257  showing  the 
hemi-celluloses  occurring  in  a  number  of  lichens,  has  been  compiled 
from  data  given  by  Karl  Muller  (11)  and  Ulander  and  Tollens  (27). 

Occurrence  and  Naturf  of  Cellulose. 

Cellulose  is  said  to  occur  in  pure  form  in  the  wall  of  the  young  plant 
cell.  With  increasing  age,  modifications  take  place  by  which  the  true 
cellulose  becomes  more  and  more  encrusted  with  lignin  and  cutin, 
two  substances  shown  by  Konig  (52),  Fiirstenberg  (39),  and  Murdfield 
(63)  to  be  almost  entirely  indigestible.  According  to  Wielen  (87)  and 
Hof meister  (43) ,  even  pure  cellulose  is  not  a  simple  substance,  but  can 
be  separated  into  soluble  and  insoluble  portions.4  Much  of  our  in- 
formation regarding  the  nature  of  cellulose  is  due  to  the  work  of 
Schulze  and  his  pupils.  Schulze  (75)  has  defined  cellulose  as  that  part 
of  the  cell  wall  giving  the  typical  cellulose  reactions,5  and  yielding 
dextrose  on  hydrolysis  with  concentrated  sulphuric  acid. 


xFor  early  literature  see  Czapek,  Biochemie  der  Pnanzen,  Vol.  I,  pp.  514-516. 
2Chitin  occurs  in  Peltigera  canina  and  Evernia  prunastre. 
3Cf.  Muller  (11)  and  Ulander  (26). 

4According  to  its  behavior  in  sodium  hydroxide  solutions,  the  quantitative  rela- 
tions depending  upon  the  source  of  the  cellulose  and  the  concentration  of  the  solu- 
tion. 

insolubility  in  dilute  acids  and  alkalies;  solubility  in  ammoniacal  copper  oxide 
solutions;  and  production  of  a  blue  color  with  iodine  and  sulphuric  acid. 


Nutrition  Investigations. 


263 


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264 


Mary  Davies  Swartz, 


CYTASES  IN  THE  VEGETABLE  KINGDOM. 

By  the  early  investigators,  Haubner  (40),  Henneberg  and  Stohman 
(41),  Kiihn,  Aronstein,  and  Schulze  (54), it  was  accepted  without  much 
question  that,  since  cellulose  disappeared  from  the  alimentary  tract  of 
herbivora,  it  is  digested  like  starch,  and  equally  valuable  as  a  nu- 
trient. But  after  Tappeiner  (78), in  1884, showed  that  cellulose  could 
be  decomposed  by  micro-organisms,  and  promulgated  his  theory  that 
this  was  the  only  way  to  account  for  the  disappearance  of  cellulose 
from  the  alimentary  canal  of  ruminants,  the  matter  fell  into  great  dis- 
pute,1 and  the  question  is  not  yet  definitely  settled  as  to  how  cellulose 
is  digested  and  what  are  the  products  of  its  digestion.  A  diligent 
search  has  been  made  for  enzymes  capable  of  attacking  it  (cytases), 
but  so  far,  such  cytases  have  been  proved  to  exist  only  in  plants  and 
lower  animals.  Many  of  these  so-called  cytases  act  upon  hemicel- 
lulose  rather  than  true  cellulose,  and  will  be  discussed  in  connection 
with  the  hemicelluloses,  though  it  is  not  always  possible  to  make  a 
sharp  distinction  between  the  two.  A  careful  review  of  the  subject 
of  cytases  in  plant  physiology  up  to  1898,  has  been  made  by  Bieder- 
mann  and  Moritz  (34),  from  which  it  appears  that  the  penetration  of 
wood  by  the  mycelia  of  moulds  is  due  to  such  cytases,  and  that  a 
powerful  cellulose-dissolving  enzyme  has  been  derived  from  Peziza 
sclerotium  by  de  Bary  (37)  and  from  another  botrytis  (presumably  a 
Peziza)  by  Ward  (84),  while  Brown  and  Morris  (36)  have  described 
cytases  existent  in  germinating  grasses  which  dissolve  their  cell  walls. 
That  this  is  anything  more  than  a  diastatic  enzyme  is  denied  by  Rei- 
nitzer  (67) ;  but  Newcombe  (64)  considers  the  assumption  of  the  iden- 
tity of  all  cell- wall  dissolving  enzymes  with  diastase  as  far  from  jus- 
tifiable. Bergmann  (32)  reports  such  cytases  in  hay  and  straw. 
Scheunert  and  Grimmer  (71),  on  the  contrary,  find  none  in  oats,  corn, 
horse-beans,  lupine  seeds,  buckwheat  or  vetch.  Thus  we  see  that 
even  in  the  case  of  plants,  these  enzymes  need  to  be  isolated  and 
identified  before  we  can  arrive  at  any  satisfactory  conclusions. 

That  cellulose  can  be  dissolved  by  bacteria  has  been  demonstrated 
for  such  forms  as  Amylobacter  butyricus,  Vibrio  regula  and  Clostridium 
polymyxa  (34).  Omelianski  (65)  has  described  two  organisms  which 
ferment  cellulose,  and  Ankersmit  (31)  finding  Omelianski's  bacteria 
on  hay,  has  studied  their  behavior  when  introduced  into  the  alimen- 
tary canal  of  the  cow  on  its  food.    He  finds  that  they  do  not  increase 


:For  a  review  of  this  discussion  cf.  Lohrisch  (56). 


Nutrition  Investigations. 


265 


in  number  during  their  passage  through  the  digestive  tract,  and  there- 
fore concludes  that  they  play  a  very  inconsiderable  role  in  the  decom- 
position of  cellulose.  According  to  Van  Iterson  (81),  certain  aerobic 
bacteria,  attacking  cellulose,  form  from  it  products  which  nourish 
other  forms  (spirilla);  certain  anaerobes  are  also  shown  to  attack 
it.  Eberlein  (38),  rinding  in  the  first  stomach  of  herbivora  Infusoria 
which  utilize  cellulose  for  food,  suggests  that  these  protozoa,  digested 
farther  along  in  the  alimentary  tract,  serve  as  means  of  transforma- 
tion of  cellulose  into  products  which  the  animal  can  digest;  but  there 
is  nothing  to  indicate  that  such  forms  occur  in  sufficient  numbers 
to  be  worthy  of  much  consideration. 

Since  1906  three  investigators  have  given  the  problem  careful  at- 
tention. Scheunert  (68)  has  concluded  from  experiments  in  vitro  that 
bacteria  play  an  exclusive  role  in  the  solution  of  crude  fiber  in  the  coe- 
cal  contents  of  horses,  swine,  and  rabbits.  He  found  that  filtered 
coecal  fluid  acted  on  cellulose  much  less  than  unfiltered  or  simply 
strained  coecal  contents.  This  is  contrary  to  the  opinion  of  Hof- 
meister  (45)  and  Holdefleiss  (48),  who  attribute  the  phenomenon  to 
the  action  of  enzymes,  and  explain  the  loss  of  power  occasioned  by 
filtering  as  due  to  the  effect  of  exposure  to  the  air  upon  the  enzymes. 
Lohrisch  (57)  has  reported  that  fresh  coecal  fluid  is  effective  in  destroy- 
ing cellulose  while  heated  fluid  is  not.  On  the  other  hand,  implanting 
the  sterilized  fluid  with  coecal  bacteria  and  protozoa  would  not  restore 
its  activity.  Coecal  fluid  kept  at  38°  C.  any  length  of  time  gradually 
lost  its  cellulose-dissolving  power,  while  that  kept  on  ice  remained 
active,  v.  Hoesslin  and  Lesser  (47)  have  attempted  to  explain  these 
apparent  contradictions,  and  conclude  from  their  own  experiments 
that  anaerobic  bacteria  are  the  most  effective  agents  in  cellulose  de- 
composition in  the  intestine.  Equal  volumes  of  non-sterilized  and 
sterilized  coecal  fluid  of  the  horse,  to  which  weighed  amounts  of  cel- 
lulose had  been  added,  were  suspended  in  sterile  physiological  salt  solu- 
tion under  practically  anaerobic  conditions  and  digested  for  periods  of 
from  9  to  35  days.  The  disappearance  of  cellulose  with  the  non-steril- 
ized coecal  fluid  amounted  to  from  55.7  per  cent  to  71.2  per  cent;  with 
sterilized  fluid,  to  from  6.2  per  cent  to  42.4  per  cent.  It  was  also  found 
that  the  addition  of  1-5  grams  of  dextrose  would  effectively  protect 
the  cellulose  from  digestion  by  the  non-sterilized  fluid,  the  bacteria 
preferring  the  more  easily  attacked  carbohydrate.  The  gases  evolved 
in  these  fermentations  were  characteristic  of  bacterial  action,  being 
chiefly  methane,  carbon  dioxide,  and  hydrogen.  The  retarding  effect 
of  exposure  to  the  air  is  explained  by  the  theory  that  anaerobes  are 


266 


Mary  Davies  Swartz, 


the  effective  agents.  So,  also,  the  fact  that  Lohrisch  was  unable  to  get 
cellulose  digestion  in  sterilized  fluid  again  inoculated  with  unsteril- 
ized  fluid  is  attributed  to  the  medium's  being  an  unfavorable  one  for 
the  development  of  these  organisms,  inasmuch  as  the  addition  of  pep- 
tones to  similar  preparations  caused  in  several  cases  an  increased  de- 
composition. It  seems  fairly  well  established,  therefore,  that  the 
action  of  the  coecal  fluid  of  the  horse  is  due  to  enzymes  of  bacterial 
origin. 

CYTASES  IN  LOWER  ANIMALS. 

There  is  no  doubt  that  cytases  occur  in  some  of  the  lower  forms  of 
animal  life.  Biedermann  and  Moritz  (34)  found  a  powerful  cellulase 
in  the  secretion  of  the  liver  of  the  common  snail  (Helix  pomatia),  and 
their  observation  was  verified  by  E.  Muller  (61),  also  by  Lohrisch  (57) 
who  reports  two  series  of  experiments  in  which  snails  fed  tender  let- 
tuce leaves  digested  from  40.1  per  cent  to  81.6  per  cent  of  the  cellulose 
present.  On  the  other  hand,  Muller  (61)  could  not  verify  Knauthe's 
report  of  a  cellulase  in  the  hepato-pancreas  of  the  carp  (50) ;  Pacault 
found  none  in  the  saliva  of  Helix  pomatia  (66) ;  and  Biedermann  none 
in  the  digestive  juice  of  the  meal  worm  (Tenebrio  molitor)  or  of  the 
cabbage  worm  (Pieris  brassica)  (34).  Biedermann  also  examined  the 
faeces  of  the  cabbage  worm  microscopically  and  found  unaltered  par- 
ticles of  leaves,  from  which  he  concluded  that  much  of  the  plant  food 
eaten  is  excreted  unchanged.  Lohrisch  (56)  has  obtained  similar  re- 
sults with  caterpillars  of  sphinx  moths  (Sphinx  euphorbiae),  not  only 
in  experiments  with  intestinal  juice  in  vitro,  but  also  in  feeding  expe- 
riments in  which  the  cellulose  was  quantitively  excreted. 

Selliere  (75-76)  has  recently  added  some  interesting  contributions 
to  this  subject,  showing  that  cotton  treated  in  various  ways;  namely, 
that  recovered  after  solution  in  Schweitzer's  reagent,  that  treated 
with  concentrated  zinc  chloride,  or  with  25  per  cent  caustic  alkali  hot 
or  cold  until  the  fibers  are  swollen,  and  subsequently  washed  with 
1  per  cent  acetic  acid  and  water,  is  attacked  by  Helix  pomatia  much 
more  readily  than  the  untreated  substance.  Subsequent  drying  of 
the  treated  cotton  diminished  its  digestibility  somewhat,  suggesting 
that  the  physical  condition  of  the  cellulose  is  a  definite  factor  in  its 
utilization.  Selliere  believes  that  only  the  more  tender  portions  of 
plant  cellulose  are  attacked  by  the  digestive  juice  of  this  snail.  It 
would  seem  that  the  previous  treatment  of  th  3  cellulose  is  a  factor  to  be 
kept  in  mind  in  the  interpretation  of  the  results  of  feeding  experiments.1 


*Cf.  the  experiments  on  cellulose  utilization  in  the  dog,  p.  263. 


N  utrition  Investigations. 


267 


CYTASES  IN  HIGHER  ANIMALS. 

There  is  at  present  no  proof  of  the  existence  of  cytases  in  any  of  the 
higher  animals.  The  literature  on  the  subject  has  been  exhaustively 
reviewed  by  Bergmann  (32),  and  Lohrisch  (55,  56,  57)  and  it  appears 
that  there  is  no  cellulase  in  the  saliva  or  pancreatic  juice  of  swine, 
horses,  cattle,  or  sheep.  The  old  observation  by  MacGillawry1  (cited 
by  Biedermann  and  Moritz(34)  that  a  cytase  can  be  extracted  from 
the  vermiform  appendix  of  the  rabbit  has  been  denied  by  Zuntz  and 
DegtiarefT  (88).  Schmulewitsch's2  statements  (also  cited  by  Bieder- 
mann and  Moritz)  are  worthless  because  he  employed  no  antiseptics. 
E.  Miiller  (61)  found  no  sugar  formed  from  the  decomposition  of  cel- 
lulose in  the  stomach  of  the  goat,  and  Lusk  (59)  observed  no  increase 
in  sugar  elimination  after  feeding  a  phlorhizinized  dog  20  grams  of 
cauliflower,  or  a  phlorhizinized  goat  10  grams  of  paper.  Lohrisch  (57) 
fed  pure  cellulose  (5-20  grams)  to  a  phlorhizinized  rabbit  and  found 
that  it  had  no  marked  influence  on  the  sugar  output,  and  no  nitrogen- 
sparing  effect.  Scheunert  (70)  has  made  further  investigation  on  the 
action  of  the  saliva  and  salivary  glands  in  sheep,  and  confirms  the 
earlier  experiments  with  the  saliva  of  this  animal.  On  the  other  hand, 
Selliere  (77)  reports  that  the  specially  treated  cellulose  mentioned 
above  is  converted  into  dextrose  by  the  intestinal  secretions  of  the 
guinea  pig  in  some  instances. 

Practically  nothing  is  known  concerning  the  way  in  which  cellulose 
disappears  from  the  alimentary  tract  of  man.  Schmidt  and  Loh- 
risch (73)  fed  pure  cellulose  to  diabetics  and  observed  a  disappearance 
averaging  77.7  per  cent,  and  no  increase  in  the  elimination  of  sugar. 
They  believe  that  most  of  it  is  absorbed  in  soluble  form  and  not  de- 
stroyed by  fermentation  in  the  intestines.  Lohrisch,  having  fed  cel- 
lulose in  various  diseases  of  the  alimentary  tract,3  calls  attention  to 
the  fact  that  in  constipation,  where  there  is  the  least  bacterial  action, 
the  utilization  of  cellulose  is  highest,  while  in  fermentation  dyspepsia, 
in  which  one  might  expect  a  marked  disappearance,  the  utilization  is 
lowest.  He  therefore  considers  the  digestion  of  cellulose  as  due  at 
least  in  part  to  enzymes. 


xArchiv  Neerland,  Vol.  XI. 

2tjber  das  Verhalten  der  Verdauungssafte  zur  Rohfaser  der  Nahrungsmittel. 
Bulletin  de  l'Academie  Imperial  de  St.  Petersburg,  1879. 
3See  results,  p.  264. 


268 


Mary  Davies  Swartz, 


Digestion  and  Utilization  of  Cellulose  by  Animals. 

The  literature  on  the  digestion  of  cellulose  up  to  1909  has  been  so 
exhaustively  reviewed  by  Lohrisch  that  it  is  unnecessary  to  enter  into 
a  detailed  discussion  of  it.  From  tables  (55)  showing  the  results  of 
all  previous  experiments  on  the  utilization  of  crude  fiber  in  herbivora, 
carnivora,  and  birds,  it  appears  that  in  the  case  of  herbivora,  especi- 
ally ruminants,  20-28  per  cent  of  the  crude  fiber  ingested  with  food 
disappears  from  the  alimentary  canal;  that  in  case  of  carnivora1  and 
birds2  there  is  no  utilization  whatever.  Lohrisch  (56)  himself  reported 
three  experiments  in  which  dogs  were  fed  pure  cellulose  and  digested 
31.1  per  cent,  37.45  per  cent  and  5.4  per  cent  respectively,  but  Scheu- 
nert  and  Lotsch  (72)  repeating  Lohrisch's  work  with  a  somewhat  dif- 
ferent method  of  determining  cellulose  found  that  the  administration 
of  40  grams  of  prepared  white  cabbage,  containing  7.37  grams  of  pure 
cellulose,  resulted  in  the  recovery  of  the  total  amount  ingested.  Cook- 
ing the  cabbage  in  bouillon  did  not  increase  its  digestibility.  They 
attribute  the  apparent  utilization  in  the  preceding  experiment  to  des- 
truction of  cellulose  by  the  reagents  used  for  its  purification.  Since 
the  publication  of  their  paper,  Lohrisch  has  repeated  his  work  with 
the  dog  (57),  and  reports  complete  recovery  of  the  cellulose  fed.  He 
explains  the  error  in  the  earlier  investigation  as  due  to  the  fact  that 
the  ingested  cellulose  was  twice  subjected  to  purification  (before  feed- 
ing and  in  faeces)  with  consequent  increase  in  percentage  of  loss, 
which  was  not  taken  into  account.  He  points  out  the  inevitable  loss 
of  some  cellulose  by  any  method  at  present  in  use  for  its  determina- 
tion, and  defends  his  own  as  sufficiently  accurate  for  all  practical  pur- 
poses if  conditions  are  carefully  observed.3 


^he  only  experiments  on  record  are  by  Voit  and  Hoffmann  on  the  dog  and  by 
von  Knieriem  on  the  hen. 

Experiments  by  Weiske  on  the  goose,  and  by  von  Knieriem  on  the  hen. 

3Lohrisch  used  the  method  of  Simon  and  Lohrisch,  in  which  the  cellulose  is  dis- 
solved by  heating  for  an  hour  on  a  water  bath  with  50  per  cent  potassium  hydroxide, 
then  adding  f  cc.  of  30  per  cent  hydrogen  peroxide,  and  digesting  from  |tof  hour 
longer  if  necessary.  The  cellulose  is  then  precipitated  by  adding  to  the  solution 
one  half  its  volume  of  96  per  cent  alcohol  and  6-7  cc.  of  concentrated  acetic  acid; 
filtered  off,  washed  with  water,  dilute  acetic  acid,  alcohol  and  ether,  dried  and 
weighed. 

Scheunert  and  Lotsch  mix  the  substance  to  be  analyzed  with  100  cc.  of  cold  water, 
add  100  grams  of  potassium  hydroxide  and  heat  for  an  hour  on  a  water  bath,  then 
filter  through  a  hard  filter  paper,  wash  the  residue  on  the  paper  with  boiling  water 
till  only  a  trace  of  alkali  remains,  transfer  it  to  a  beaker  and  thence  to  a  weighed 


'  Nutrition  Investigations. 


269 


Cellulose  digestion  in  the  dog  has  been  almost  simultaneously  stud- 
ied by  v.  Hoesslin  (46).  Two  dogs  on  a  meat-fat  diet  to  which  was 
added  daily  2  grams  of  specially  prepared  white  cabbage  (containing 
63.25  per  cent  of  pure  cellulose),  for  five  periods  of  five  days  each, 
excreted  on  the  average  99.7  per  cent  and  94.5  per  cent  respectively. 
This  long  experiment  is  significant  as  showing  no  adaptation  of  the 
digestive  glands  to  the  type  of  food.  By  these  independent  workers 
it  seems  now  well  established  that  the  dog  is  unable  to  utilize  cellulose. 

Hoffmann  (42)  has  just  published  the  results  of  some  investigations 
on  the  influence  of  cellulose  on  the  nitrogen  balance  and  on  phlo- 
rhizin-diabetes  in  the  rabbit,  from  which  it  appears  that  after  inges- 
tion there  is  no  increase  of  sugar  excretion,  and  no  glycogen  formation, 
yet  he  thinks  that  cellulose  and  hemicelluloses  have  a  favorable  influ- 
ence in  phlorhizin-diabetes.1  It  seems  to  follow  from  this,  that  even 
in  case  of  herbivora  cellulose  is  not  utilized  in  the  manner  customary 
for  starch  and  sugar. 

DIGESTION  AND  UTILIZATION  OF  CELLULOSE  BY  MAN. 

A  similar  tabulation  of  results  of  feeding  experiments  on  man,  shows 
that  cellulose  is  not  so  well  utilized  as  by  herbivora,  but  does  disap- 
pear in  appreciable  amounts.  With  one  exception,  the  cellulose  in 
all  these  experiments  was  administered  as  crude  fiber.  Hofmeister 
(43)  fed  pure  cellulose  and  reported  75.7  per  cent  soluble  cellulose  and 
5.6  per  cent  insoluble  cellulose  digested.  Konig  and  Reinhardt  (53) 
added  to  a  diet  rich  in  protein  and  fat,  but  free  from  cellulose,  in  sev- 
eral experiments,  green  peas  and  ripe  shelled  peas,  red  cabbage,  wThite 


filter,  on  which  it  is  washed  successively  with  hot  water,  dilute  acetic  acid,  hot  water, 
alcohol  and  ether,  and  finally  weighed. 

Scheunert  and  Lotsch  claim  that  by  Lohrisch's  method  the  cellulose  is  altered  in 
character,  and  as  much  as  40  per  cent  lost  in  the  process;  and  that  subsequent  treat- 
ment of  the  recovered  material  causes  an  even  greater  per  cent  of  loss,  while  by  their 
method  the  loss  in  the  first  case  is  not  over  6.8  per  cent,  and  that  in  the  second  case 
even  less. 

For  the  details  of  this  controversy  over  method  see  the  following :  Simon  and  Loh- 
risch;  Zeitschrift  fur  physiologische  Chemie,  Vol.  42,  p.  55,  (1904).  Scheunert; 
Berliner  tierarztliche  Wochenschrif t,  No.  47,  p.  826,  (1909) .  Scheunert  and  Lotsch; 
Ibid.,  p.  867,  (1909);  also  Biochemische  Zeitschrift,  Vol.  20,  p.  10,  (1909);  and 
Zeitschrift  fur  physiologische  Chemie,  Vol.  65,  p.  219,  (1910).  Scheunert  and 
Grimmer;  Berliner  tierarztliche  Wochenschrif t,  No.  48,  p.  152,  (1910).  Lohrisch; 
Zeitschrift  fur  physiologische  Chemie,  Vol.  69,  p.  143,  (1910). 

Unfortunately  the  original  paper  was  not  accessible. 


270 


Mary  Davies  Swartz, 


beans,  graham  and  soldiers'  bread  and  found  30.27  per  cent  to  76.79 
per  cent  of  the  added  cellulose  digested.  Lohrisch  (55)  finds  that  the 
cellulose  of  a  common  vegetable  diet  disappears  from  the  alimentary 
tract  in  large  amounts,  the  actual  quantity  varying  with  the  age, 
source  and  tenderness  of  the  cellulose.  Thus  he  finds  that  for  normal 
individuals,  of  cellulose  from  lentils,  45  per  cent  is  digestible;  from 
kohlrabi,  79.1  per  cent;  from  white  cabbage,  100  per  cent.  Under 


abnormal  conditions  in  the  digestive  tract,  he  has  obtained  the  fol- 

lowing results: 

CONDITION. 

CELLULOSE  UTILIZATION  IN  PER  CENT. 

Normal  

57.9 

Chronic  Constipation  

81.4 

Fermentation  Dyspepsia  

37.8 

Gastrogenic  Diarrhea  

29.5 

Fatty  Faeces  in  Icterus  

27.8 

Fatty  Faeces  in  Disease  of  Pancreas  

20.9 

According  to  Lohrisch,  two  diabetics  on  a  cellulose-free  diet,  to 
which  white  cabbage  was  added  in  quantities  to  yield  about  6  per 
cent  of  cellulose  per  day,  digested  68.6  per  cent  and  84.5  per  cent 
respectively,  without  increased  output  of  sugar  in  the  urine. 

Since  the  only  way  to  determine  definitely  the  energy  value  to  the 
organism  of  such  amounts  of  cellulose  as  are  absorbed,  is  by  means 
of  respiration  experiments,  Lohrisch  (57)  has  performed  such  an  expe- 
riment on  man,  using  the  Zuntz-Geppert  apparatus.  In  fasting,  the 
respiratory  quotient  averages  about  0.76.  After  ingestion  of  carbo- 
hydrates such  as  starch,  it  rises  gradually  in  two  to  three  hours,  to 
0.9-1.0,  and  when  the  carbohydrate  has  been  consumed,  sinks  again 
to  a  lower  level.  Since  the  respiratory  quotient  for  fat  is  0.7  and 
for  protein  about  0.8,  it  is  possible  to  determine  in  this  way  to  what 
extent  the  carbohydrate  replaces  protein  and  fat  in  metabolism. 
Hence  if  cellulose  is  absorbed  and  oxidized  as  a  carbohydrate,  the  res- 
piratory quotient  should  rise.  If  it  is  decomposed  by  bacteria,  the 
respiratory  quotient  should  not  rise,  since  the  theoretical  respiratory 
quotient  for  fatty  acids,  such  as  butyric  and  acetic,  is,  according  to 
Munk  (62)  and  Mallevre  (60),  0.6  and  0.5  respectively.  Now  Loh- 
risch, feeding  a  man  moist  cellulose  equivalent  to  73.6  grams  of  dry 
substance,  of  which  25  per  cent  was  digested  (18.5  grams)  obtained  the 
following  results : 


Nutrition  Investigations . 


•NOIX 

-saoNi  asonii'iao 

aO  0NINNI03S  aaXiV 

1 

•NOixonaoHd  zoo 

§                                  H|N                       H|lN   H|N  iH|N 

b              1    1    1    1    1  +  1  + 

•NOIXdHJlSNOD  «0 

»i  He* 
S                          him  h|c*              CD  O 

H(NlOCO(OHH^ 

+    1    +  +  +  +  +  + 

•axnNiH 
aaa  NOixonaoaa  zoo 

ccm. 

156.34 
146.50 
151.42 
142 . 79 
143.82 
150.16 
147.81 
141.36 
157.78 
146.26 
159.23 

•axaNm 

H3d  NOIXdHXlSNOO  &0 

ccm. 

194.37 
189.89 
192.13 
194.47 
187.38 
202.69 
203.49 
203.26 
223.82 
211.37 
219.53 

"0  'H 

oot>t^i>^t^t^r^t^©t^ 
ooooooooooo 

•Noixonaoaa  zoo 

Per  cent. 

3.33 
3.68 

3.37 
3.40 
3.23 
3.10 
3.22 
3.10 
3.01 
3.01 

*NOIXJPin.SNOO  ^00 

Per  cent. 
4.14 
4.77 

4.59 
4.43 
4.36 
4.27 
4.63 
4.44 
4.35 
4.15 

'axnNrre  Had 
aaavKNi  aimoA 

•  lOi-h         NO05  00OHOO 
§0300  MW^tOOJ^iOOl 
S  O  OS  (NCNCONCOOOOIN 
U          CO  T^TjH^-H^TiHlOTtltO 

•saxn 

•"IxiJ'V    Ix±  JLiN.  killU  1  a.  cl  Cl 

-xa    ao  Noixvana 

•XNararaad 
-xa   ao  ONifjNioaa 

v."  M  iO  rHt^cOOOOOCMCO 
tq  CO  N  OH^HN^CDN 

•xNara 
-raaaxa  ao  naaiinN 

,-H  <N  >M^»OCDNOOOJO 

272 


Mary  Davies  Swartz, 


The  respiratory  quotient  attains  its  highest  value  in  the  fourth 
hour,  instead  of  the  second  or  third,  showing  that  cellulose  is  absorbed 
more  slowly  than  starch.  The  rise  is  too  slight  to  indicate  that  cellu- 
lose exercises  any  considerable  protein-  or  fat-sparing  effect.  It 
is  unfortunate  that  the  amount  of  cellulose  absorbed  was  so  small. 
It  is  striking  that  the  02-consumption  decreases  at  the  very  time  that 
the  respiratory  quotient  rises,  and  the  C02-production  scarcely  in- 
creases. Lohrisch  interprets  this  as  indicating  that  the  increased  02- 
consumption  required  for  oxidation  of  the  cellulose  is  compensated  by 
a  sparing  of  protein  and  fat.  The  differences  seem  too  small  to  draw 
any  satisfactory  conclusions  as  to  the  energy  value  of  cellulose.  The 
low  respiratory  quotient  in  the  later  hours  of  the  experiment,  together 
with  the  increased  Oo-consumption,  indicates  the  utilization  of  some 
of  the  cellulose  in  the  form  of  fatty  acids.  We  must  bear  in  mind 
that  no  formation  of  sugar  or  glycogen  from  cellulose,  in  men  or  ani- 
mals, has  been  demonstrated.  Further  investigations  would  seem  to 
be  necessary  before  we  can  agree  with  Lohrisch  in  saying,  "  Wir  wissen, 
dass  Cellulose  und  Hemi-cellulosen  votn  Menschen  reichlich  verdaut 
werden,  wir  haben  alien  Grund  anzunehmen,  dass  ihre  Verdauung  nach 
Analogie  der  Starke  ablduj I  .  .  .  Die  resorbirten  Mengen  werden  im 
menschlichen  Organismus  vollstdndig  verbrannt.  Dabei  wird  Eiweiss 
und  Fett  von  der  Verbrennung  geschiitzt."  In  any  event,  the  quanti- 
ties of  cellulose  which  the  alimentary  tract  of  man  is  capable  of  ab- 
sorbing are,  apparently,  too  small  for  it  to  play  a  role  of  any  impor- 
tance in  the  diet  of  a  normal  individual. 

Occurrence  and  Nature  of  Pentosans. 

The  anhydrides  of  the  5-carbon  sugars  are  collectively  designated 
as  pentosans.  These  are  not  reported  to  occur  in  the  animal  kingdom, 
but  the  pentose  sugars  are  found  forming  a  part  of  the  nucleic  acid 
radical  of  the  nucleo-protein  molecule.  In  the  vegetable  kingdom, 
pentosans  are  very  widely  distributed,  as  has  been  shown  by  many 
investigators,  especially  Tollens  and  his  pupils.1  They  occur  in  all 
kinds  of  plants,  from  the  lowest  to  the  highest,  and  are  limited  to  no 


1  Tollens,  Landw.  Vers.,  V.  39,  p.  401,  (1891);  Tollens,  Jour.  f.  Landw.,  Vol.  44, 
p.  171  (1896). 

For  an  exhaustive  review  of  the  literature  on  the  occurrence  of  the  pentosans 
see  v.  Lippmann,  Chemie  der  Zuckerarten,  3rd  Edition,  Vol.  I,  pp.  44-60;  116-123; 
and  Czapek,  Biochemie  der  Pflanzen,  Vol.  I,  pp.  537-545  (1905). 


Nutrition  Investigations . 


273 


particular  organ  or  tissue,  being  found  abundantly  in  roots,  stems, 
leaves  or  seeds. 

In  regard  to  solubility  in  water,  pentosans  show  all  possible  varia- 
tions. De  Chalmot  (108)  found  them  present  in  the  watery  extract  of 
the  leaves  of  many  plants;  Winterstein  (167)  in  the  somewhat  mucila- 
ginous hot  water  extract  of  the  seeds  of  Tropaeolum  majus;  Schulze  (146) , 
in  both  soluble  and  insoluble  form  in  the  cotyledons  and  endosperms 
of  the  seeds  of  Lupinus  luteus  and  other  legumes,  where  they  are  doubt- 
less stored  as  reserve  material  for  the  growing  plant;  and  in  the  cell 
walls  of  the  mature  plants,  where  in  most  cases  they  approach  true  cel- 
lulose in  character.  It  is  difficult  to  differentiate  these  highly  resis- 
tant pentosans  of  the  cell  wall,  which  are  commonly  included  in  the 
term  crude  fiber,  from  the  ligno-celluloses  and  oxycelluloses  also 
found  there,  which  as  Cross,  Bevan  and  Beadle  (104)  have  shown,1 
are  like  true  pentosans  in  yielding  furfurol  on  distillation  with  dilute 
hydrochloric  acid.  Besides  hemicelluloses  yielding  pentoses  (xylose 
and  arabinose)  exclusively,  occur  many  yielding  also  methyl-pentoses 
(fucose,  rhamnose) .  These  yield  on  distillation  with  dilute  hydrochloric 
acid,  methyl-furfurol,  which  is  precipitated  by  phloroglucin,  and  hence 
included  in  quantitative  estimations  of  pentosans  by  the  method  of 
Tollens  andKrober  (121).  The  distribution  of  methyl-pentosans  has 
been  studied  especially  by  Tollens  and  his  pupils.  Japanese  "Nori" 
(Porphyra  laciniata,  Laminaria,  and  other  seaweeds)  (129),  tragacanth 
and  many  other  gums  (163)  contain  fucosan.  Rhamnose  occurs  also 
widely  distributed  in  the  plant  kingdom,  but  more  frequently  in  the 
form  of  a  glucoside.  Rohmann  (134)  reports  a  rhamnosan  in  Ulva 
lactuca. 

It  is  a  very  common  thing  to  find  pentosans  and  hexosans  occurring 
together.  In  fact,  it  is  absolutely  impossible,  in  treating  of  hemicellu- 
loses, to  draw  any  sharp  dividing  lines,  for  they  are  not  only  intimately 
associated,  but  frequently  chemically  combined.  Schulze  (146)  has 
given  the  name  paragalactan  to  the  carbohydrate  yielding  arabinose 
and  galactose,  which  occurs  in  the  seeds  of  many  legumes.  Winter- 
stein (167)  finds  galacto-xylan  in  the  water  extract  of  Tropaeolum 
majus,  and  numerous  other  examples  of  such  combinations  might  be 
cited. 

A  class  of  substances  to  which  has  been  given  a  distinctive  name 
because  of  their  peculiar  gelatinizing  property,  is  the  Pectins.  As 
Czapek2  remarks,  "It  is  uncertain  whether  they  form  a  definite 


:For  further  details  see  v.  Lippmann;  Chemie  der  Zuckerarten,  Vol.  I,  pp.  160-169^ 
2Die  Pektin-Substanzen;  Czapek,  Biochemie  der  Pflanzen,  Vol.  I,  p.  545. 


274 


Mary  Dames  Swartz, 


class  of  cell  wall  substances,  or  whether  they  should  be  classified 
as  'hemicelluloses'  or  'pentosans.'  "  In  1868,  Scheibler  (141)  found  a 
sugar  which  he  called  pectinose,  but  which  was  later  shown  to  be  ara- 
binose  (142).  In  1875,  Reichardt  (132)  obtained  a  pectin  body  from 
carrots  and  beets,  which  he  called  'pararabin,'  expressing  the  view 
that  pectins  should  hardly  be  considered  as  a  special  class  of  carbo- 
hydrates. Tromp  de  Haas  and  Tollens  (160)  have  found  from  numer- 
ous analyses,  that  the  pectins  do  not  differ  from  other  carbohydrates 
in  their  relative  proportions  of  hydrogen  and  oxygen  so  much  as  earlier 
workers  supposed,  and  hence  they  may  be  classified  with  other  hemi- 
celluloses according  to  the  products  of  their  hydrolysis  (pentoses; 
galactose  and  other  hexoses).  Cross  (106)  believes  them  to  be  allied 
to  the  ligno-celluloses.  The  whole  matter  is  still  in  a  state  of  uncer- 
tainty. Herzfeld  (116)  has  shown  that  arabinose  can  be  obtained 
from  most  pectins,  and  consequently  they  have  been  included  among 
the  pentosans,  though  from  the  frequency  with  which  they  yield  ga- 
lactose, they  might  equally  well  be  discussed  with  the  galactans.  Ac- 
cording to  Czapek  while  pectins  occur  frequently  in  phanerogams, 
ferns  and  mosses,  their  presence  in  algae  is  doubtful,  although  it  is 
possible  that  soluble  carbohydrates  of  algae  yielding  arabinose  or  ga- 
lactose are  closely  related  to  the  pectins  of  other  plants.1 

Role  of  the  Pentosans  in  Plant  Physiology. 

Comparatively  little  is  known  of  the  role  of  pentosans  in  plant  phys- 
iology. De  Chalmot's  (108)  observation  that  they  decrease  in  quan- 
tity in  seeds  —  peas  and  corn  —  during  germination,  and  reappear 
in  the  stems  and  roots  of  the  growing  plant,  would  seem  to  indicate 
that  they  form  a  part  of  the  reserve  material  in  the  seed;  but  Schone 
and  Tollens  (145),  finding  no  diminution  in  the  amount  of  pentosans 
in  grains  during  germination,  but  rather  a  slight  increase,  declare  that 
they  do  not  belong  to  the  reserve-stuff  of  the  seed;  so  the  question  may 
be  regarded  as  still  unsettled.  Changes  in  the  relative  amounts  of 
pentosan  in  plants  at  different  stages  of  growth,  studied  by  Cross, 
Bevan  and  Smith  (105),  Gotze  and  Pfeiffer  (113),  Calabresi  (98),  and 
others,  show  that  the  increase  of  pentosans  runs  parallel  to  the  forma- 
tion of  the  skeletal  substance;  and  have  led  to  the  idea  that  they  arise 
through  the  transformation  of  a  part  of  the  cellulose,  and  along  with 
lignin  and  cutin,  take  part  in  wood  formation.    Ravenna  and  Cereser 


JCf.  also  Bigelow,  Gore,  and  Howard  (92). 


Nutrition  I  nvestigations. 


275 


(131)  find  in  the  case  of  dwarf  beans  that  when  the  food  is  wholly  dex- 
trose administered  to  the  leaves,  pentosans  increase  greatly,  especially 
in  the  light,  and  that  when  the  functioning  of  chlorophyll  is  prevented 
for  long  periods  the  amount  of  pentosans  decreases.  They  conclude 
that  the  simple  sugars  exert  a  preponderating  influence  in  pentosan 
formation,  and  that  these  serve  as  a  reserve  material  when  the  plant 
has  exhausted  its  more  readily  available  food  materials. 

PENTOSANASES  IN  THE  VEGETABLE  KINGDOM. 

Our  knowledge  of  enzymes  inverting  pentosans  is  meager,  and  rather 
indefinite.  The  action  of  such  forms  as  Hymenomycetes  upon  wood 
seems  to  be  of  chemical  nature.  At  any  rate  it  is  evident  (107-146) 
that  they  are  able  to  utilize  xylan.  Bourquelot  and  Herissey  (95)  have 
isolated  an  enzyme  from  malt  diastase  which  produces  reducing  sugar 
from  pectins,  and  call  it  pectinase.  This  is  not  to  be  confused  with 
the  so-called  pectase  which  causes  the  coagulation  of  pectin  bodies. 
Bigelow,  Gore  and  Howard  (92)  also  find  that  the  enzymes  of  Asper- 
gillus partially  hydrolyze  the  pectin  of  gentian  root.  According  to 
Harrison  (114),  Bacillus  oleraceoe  produces  a  cytase  capable  of  dissolv- 
ing the  cell  walls  of  potatoes,  turnips,  cauliflower  and  allied  plants, 
which  acts  particularly  on  the  middle  lamella,  the  supposed  seat  of 
pectin.1  The  latter  is  not  an  inverting  enzyme.  In  Persian  Berries 
(Rhamnus)  (162),  in  Penicillium  glaucum,  and  Botrytis  cinerea  (90), 
an  enzyme  (rhamnase)  has  been  found  which  splits  off  rhamnose  from 
some  of  its  glucosides  (rhamnetin  and  rhamnazin).  An  early  observa- 
tion of  the  presence  of  rhamnase  in  the  rutin  of  garden  rue  was  made 
by  Borntrager  (94).  That  some  of  the  so-called  cytases  described 
under  cellulose2  may  act  on  pentosans  seems  possible,  but  there  is  no 
direct  evidence  that  such  is  the  case.  On  the  contrary,  Cross  and 
Bevan  (105)  believe  that  pentosans  once  formed  in  the  plant,  remain 
thenceforth  unaltered. 

Tollens  and  Glaubitz  (159)  assert  that  the  pentosans  do  not  undergo 
lactic  or  butyric  acid  fermentation,  and  are  otherwise  unaffected  by 
yeast,  as  has  also  been  shown  by  Lintner  and  Dull  (125) .  The  pento- 
sans are  very  resistant  toward  the  action  of  bacteria.  Slowtzoff  (154) 
found  that  a  small  amount  of  pure  xylan  in  a  putrefying  mixture, 


xCi.  Czapek,  Biochemie  der  Pflanzen. 

2Cf.  Biedermann  and  Moritz  (34),  Brown  (35),  Brown  and  Morris  (36),  Berg- 
mann  (32),  Griiss  (184),  Newcombe  (64). 


276 


Mary  Davies  Swartz, 


kept  at  a  temperature  of  40°  C,  did  not  entirely  disappear  from  the 
solution  before  the  ninth  or  tenth  day.  Two  widely  distributed  fer- 
menting agents  acting  on  hemicellulose  {Bacillus  aster os poms  Arth. 
Meyer,  and  Bacillus  clostridieforme,  Burri  and  Ankersmit),  studied  b}' 
Ankersmit  (89),  are  said  by  him  to  occur  in  insufficient  numbers  to 
make  their  activity  of  any  significance  in  the  alimentary  canal  of  the 
cow. 

PENTOSANASES  IN  LOWER  ANIMALS. 

Extensive  investigations  regarding  the  occurrence  of  pentosan- 
splitting  enzymes  in  lower  animals,  have  been  made  by  Selliere  since 
1905.  The  secretion  of  the  hepato-pancreas  of  the  common  snail 
(Helix  pomatia)  not  only  digests  cellulose  in  vitro,1  but  also  xylan,  ac- 
cording to  this  writer  (148).  In  feeding  experiments,  analyses  of  the 
food  (oak  wood)  and  excreta  of  these  xylophages  showed  a  higher  per- 
centage of  xylan  in  the  former  than  in  the  latter  (149).  Hence  xylan 
must  have  been  digested.  In  1907,  he  showed  that  pentoses  were 
actually  liberated  and  absorbed,  by  testing  the  blood  of  these  snails, 
which  gave  the  phloroglucin  reaction  (151).  That  sugar  can  be  found 
in  their  blood  is  denied  by  Couvreur  and  Bellion  (99), but  this  Selliere 
attributes  to  the  fact  that  the  sugar  content  is  much  less  than  in 
higher  animals,  and  hence  has  been  entirely  overlooked.  - 

Xylanase  also  occurs  in  other  species  of  snail  (150)  such  as  Helix 
aspera  Miill.,  Helix  nemoralis  L.,  Limax  arborum  Bouch.,  Limax 
variegatus  Drap.,  Arion  rufus  L.,  Patella  vulgata  L.,  Littorina  lit'.orea  L., 
Littorina  littoralis  L.,  and  in  a  representative  of  the  Coleoptera,  Phy- 
tnatodes  variabilis  L.  The  presence  of  a  xylanase  in  Patella  vulgata 
and  the  Littorinae  is  especially  significant,  as  their  food  consists  in 
pentosan-rich  algae.  Selliere  (150)  and  Pacault  (130)  have  independ- 
ently discovered  a  xylanase  in  the  salivary  glands  of  Helix  pomatia. 
According  to  Rohmann(134),  Aplysia,  which  subsist  largely  upon 
Viva  lactuca,  do  not,  digest  the  soluble  methyl-pen tosan  (rhamnosan) 
present  in  this  alga.  He  finds  this  carbohydrate  present  in  the  glands 
of  the  midgut,  but  regards  it  as  a  food  residue. 

PENTOSANASES  IN  HIGHER  ANIMALS. 

There  have  been  only  a  few  investigations  as  to  the  presence  in 
higher  animals  of  enzymes  hydrolyzing  pentosans.    Slowtzoff  (154) 


lCi.  Biedermann  and  Moritz  (34). 


Nutrition  Investigations. 


277 


found  that  pure  xylan  was  not  digested  by  saliva,  gastric  or  pancre- 
atic juice,  but  could  be  gradually  hydrolyzed  (in  two  or  three  days)  by 
0.2  per  cent  hydrochloric  acid.  Bergmann  (91)  digested  pure  xylan 
with  extracts  of  the  intestines  of  many  animals  (hen,  goose,  guinea- 
pig,  sheep, ox,  horse),  and  of  the  vermiform  appendix  of  rabbits,  but  in 
no  case  found  a  xylanase.  These  experiments  were  performed  with 
suitable  antiseptics  and  controls  in  all  cases.  An  old  experiment  by 
Fudakowski  (112),  attributing  an  inverting  action  upon  gum  arabic 
to  pepsin,  and  another  by  Schmulewitsch  (144),  attributing  such  an 
action  upon  crude  fiber  to  pancreatin,must  be  disregarded,  as  no  anti- 
septics whatever  seem  to  have  been  used.  According  to  Selliere  (152), 
neither  the  pancreatic  juice  of  rabbits,  nor  a  mixture  pancreatic  and  in- 
testinal juices,  will  hydrolyze  xylan.  Negative  results  were  also  ob- 
tained by  him  with  macerated  intestines  of  these  animals.  On  the  other 
hand,  chloroform  extracts  of  the  intestinal  contents  of  rabbits  and 
guinea-pigs  fed  fresh  hay  and  bread,  produced  pentoses  in  a  5  per  cent 
xylan  solution  after  48  hours  digestion  at  37  degrees  C,  while  negative 
results  were  obtained  with  boiled  controls.  This  indicates  that  the 
enzymes  causing  hydrolysis  were  of  bacterial  origin,  a  conclusion  sub- 
stantiated by  later  work  of  the  same  author  (153).  No  xylanase  was 
detected  in  the  excreta  of  carnivora  such  as  the  lion,  panther,  and  wolf. 
From  a  centrifugalized  extract  of  human  faeces  and  soluble  xylan,  di- 
gested under  aseptic  conditions,  xylose  was  obtained  after  15-20  hours; 
but  in  meconium  of  calves  and  human  beings  in  which  bacteria  were 
absent  no  xylanase  could  be  found,  although  the  intestinal  glands  were 
functioning.  McCollum  and  Brannon  (126)  have  shown  that  in  the 
case  of  the  cow  intestinal  bacteria  destroy  pentosans  under  anaerobic 
conditions,  the  degree  of  destruction  varying  with  the  kind  of  plant. 
Corn,  wheat  and  oat  feeds  were  incubated  with  fecal  bacteria  of  this 
animal,  and  digestions  continued  14  days  in  atmospheres  both  of  car- 
bon dioxide  and  hydrogen,  with  the  following  average  results: 


MATERIAL 

Com  Fodder  

Corn  Fodder  

Wheat  Straw.  .  .  . 

Wheat  Straw  

Oat  Straw  

Oat  Straw  


ATMOSPHERE. 


PER  CENT  OF  PENTOSANS  DISAPPEARING. 


co2 

H 

C02 
H 

C02 
H 


51.78 
76.13 
28.09 
37.99 
30.66 
54.00 


From  this  review  it  is  evident  that  the  presence  of  pentosanases  in 
the  higher  animals  has  not  yet  been  demonstrated. 


278 


Mary  Davies  Swartz, 


DIGESTION  AND  UTILIZATION  OF  PENTOSANS  BY  ANIMALS. 

In  the  case  of  men  and  animals  subsisting  on  a  mixed  diet,  the  hex- 
oses  and  their  derivatives  so  overbalance  the  pentosans,  under  normal 
conditions,  that  the  utilization  of  the  latter  is  a  question  of  theo- 
retical rather  than  of  practical  importance.  But  in  the  case  of  herbi- 
vora,  limited  to  a  diet  in  which  pentosans  occur  in  considerable 
amounts,  the  extent  of  pentosan  utilization  becomes  a  question  of 
economic  importance.  It  is  not  surprising  to  find,  therefore,  that 
since  the  development  of  satisfactory  methods  of  quantitative  deter- 
mination, a  considerable  number  of  investigations  have  been  made 
upon  such  utilization  by  animals.  The  results  of  these  experiments 
are  shown  in  tables  on  pages  274  and  275. 

The  results  in  these  experiments  were  obtained  by  analysis  of  food 
and  faeces.  Lindsey  (123)  Gotze  and  Pfeiffer  (113)  and  Tollens  (157) 
found  no  measurable  amount  of  pentoses  or  pentosans  excreted  in  the 
urine  of  sheep,  but  Neuberg  and  Wohlgemuth  (128)  state  that  pento- 
sans always  occur  in  the  urine  of  rabbits,  only  disappearing  when  the 
vegetable  diet  is  compensated  by  pentose-free  material.  They  report 
that  9  per  cent  of  soluble  araban  (cherry  gum)  fed  to  rabbits  was  ex- 
creted in  the  urine.  Slowtzoff  (154)  found  1.4-4.5  per  cent  of  xylan  in 
the  urine  of  rabbits,  but  no  reducing  sugar.  He  also  found  that  if 
the  animal  were  killed  shortly  after  xylan  feeding,  xylan  could  be  de- 
tected in  blood,  liver  and  muscles.  Hence  xylan  must  have  been  ab- 
sorbed from  the  digestive  tract. 

The  feeding  experiments  show  that  herbivora  digest,  on  the  aver- 
age, 55-60  per  cent  of  the  pentosans  in  their  diet,  but  since  no  animal 
enzymes  hydrolyzing  pentosans  have  been  demonstrated,  and  there 
is  always  the  possibility  of  bacterial  decomposition  in  the  intestines, 
the  most  conclusive  experiments  as  to  the  actual  nutritive  value  are 
those  of  Kellner  (118)  with  the  respiration  calorimeter.  From  the 
slight  difference  in  loss  of  potential  energy,  when  the  furfurol-yielding 
rye  straw  preparation  was  substituted  for  starch,  he  concludes  that 
furfurol-yielding  substances  participate  in  the  formation  of  fat  in  the 
animal  body. 

DIGESTION  AND  UTILIZATION  OF  PENTOSANS  BY  MAN. 

We  have  seen  that  pentosans  can  be  digested  by  herbivora  to  a 
considerable  extent.  Can  they  be  digested  by  man?  The  only 
feeding  experiments  on  record  are  by  Konig  and  Reinhardt  (120). 


Nutrition  Investigations . 


279 


In  1902,  they  conducted  researches  on  two  men  whose  main  diet  con- 
sisted of  meat  and  butter  or  other  fat,  and  beer;  to  this,  in  the  various 
experiments,  were  added  respectively  (along  with  sugar,  butter,  beef 
extract,  etc.,  used  in  preparing  them)  the  following  substances: 

Experiment  I.  Green  Peas.  Experiment  II.  Ripe  Shelled  Peas. 
Experiment  III.  Red  Cabbage.  Experiment  IV.  Canned  White 
Beans.  Experiment  V.  Soldiers'  Bread.  Experiment  VI.  Graham 
Bread. 

From  analyses  of  food  and  faeces  the  following  results  were  obtained: 


TOTAL  PENTOSANS  IN  GRAMS. 

EXP.  I. 

ii. 

in. 

IV. 

V. 

VI. 

15.55 

23.15 

14.01 

12.80 

52.64 

41.26 

0.79 

0.59 

0.70 

1.12 

8.66 

4.06 

Per  cent  not  utilized,  estimating  Pen- 

tosans in  Beer  as  unutilized  

5.08 

2.55 

5.0 

8.75 

16.45 

9.84 

7.47 

3.24 

7.75 

14.32 

20.24 

12.97 

Hence  we  see  that  of  the  total  pentosans  in  the  diet  3.24-20.24  per 
cent  were  excreted.  Only  a  little  furfurol-yielding  substance  was 
found  in  the  urine.  From  the  small  percentage  recovered  in  these 
experiments,  Konig  and  Reinhardt  (120)  conclude  that  the  pentosans 
are  to  a  high  degree  utilized  by  man,  but  they  take  no  account  of  pos- 
sible destruction  by  bacteria.1 

Since  pentosans  do  disappear  from  the  alimentary  tract  of  men  and 
animals,  it  behooves  us  to  consider  whether,  on  the  assumption  that 
they  are  hydrolyzed  like  starch,  the  pentose  sugars  so  produced  are 
as  well  utilized  as  dextrose.  Konig  and  Reinhardt  (120)  found  some 
furfurol-yielding  substance  in  the  urine,  and  Blumenthal  (93)  observes 
that  after  eating  huckleberries,  cherries  and  prunes,  pentosans  are 
excreted,  but  no  reducing  sugar.  Cominotti  (100)  finds  pentoses  ab- 
sent from  the  urine  of  man  on  a  meat  diet,  but  always  present  on  a 
mixed  diet.  He  agrees  with  Konig  and  Reinhardt  that  the  output  in 
the  urine  is  small  compared  with  the  amount  of  pentosans  in  the  food, 
and  proposes  to  investigate  the  possibility  of  glycogen  formation  from 
pentosans. 

The  behavior  of  pentoses  in  the  body  has  been  exhaustively  reviewed 
by  Neuberg(127).2   It  appears  from  the  work  of  Cremer  (102,  103), 

1  Cramer  (101)  has  shown  (according  to  a  recent  review,  the  original  paper  was 
not  accessible)  that  bacteria  are  essential  to  hemicellulose  transformation. 

2For  a  recent  discussion  of  the  absorption  and  utilization  of  pentoses  see  A.  Mag- 
nus-Levy, Oppenheimer's  Handbuch  der  Biochemie  der  Menschen  und  der  Tiere, 
1909,  Vol.  IV,  pp.  395-407. 


280 


Mary  Dames  Swartz, 


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282 


Mary  Davies  Swartz, 


Ebstein  (109),  Frantze  (111),  Neuberg  and  Wohlgemuth  (128),  Sal- 
kowski  (137),  v.  Jacksch  (117),Lindemann  and  May  (122),  Brasch  (96) 
and  others,  that  the  pentoses  and  methyl-pentoses  (rhamnose)  are  ex- 
creted more  readily  than  the  hexoses;  that  they  exert  an  unfavorable 
effect  in  diabetes;  and  that  there  is  no  evidence  of  their  acting  as  gly- 
cogen-formers  in  man.  Consequently,  even  if  further  experiments 
justify  Konig  and  Reinhardt's  conclusions,  the  pentosans  must  appar- 
ently still  play  a  very  small  part  in  the  nutrition  of  man. 

Occurrence  and  Nature  of  Galactans. 

Next  to  the  pentosans,  no  hemicelluloses  seem  to  be  so  widely  dis- 
tributed as  the  galactans;  both  occur  together  in  the  plant  cell,  and 
often  in  a  more  or  less  intimate  chemical  combination.  The  pure 
galactans,  i.e.,  those  yielding  exclusively  galactose  upon  hydrolysis, 
have  been  differentiated  into  several  classes,  chiefly  by  differences  in 
s  jlubility  or  specific  rotation,  namely: 

1.  a-galactan,  so  named  by  Miintz  (199),  the  first  to  identify 
galactan  as  an  anhydride  of  galactose;  it  composes  42  per  cent  of 
luzerne  seeds  and  occurs  also  in  beans,  barley,  and  malt. 

2.  jS-galactan,  isolated  from  the  lime  residues  in  the  sugar  beet 
industry  by  Lippmann  (192). 

3.  7-galactan,  first  isolated  from  Chinese  moss  (Sphaerococcus 
lichenoides)  by  Payen  (262),  in  1859,  and  by  him  called  "gelose." 
He  also  identified  it  in  agar-agar1  (Gelidium  corneum)  and  other  algae. 
The  carbohydrates  of  agar-agar  were  again  studied  by  Reichardt  in 
1876,  who  obtained  a  substance  of  the  formula  C12H22O11  and  con- 
sidered it  identical  with  the  "pararabin"  which  he  found  in  carrots 
and  beets.2  In  1881  and  1882,  Greenish  (180,  181)  investigated  the 
carbohydrates  of  Fucus  amylaceus  (Ceylon  agar-agar)  and  obtained 
on  hydrolysis  a  sugar-yielding  mucic  acid  (galactose).  From  Sphaero- 
coccus lichenoides  he  also  obtained  a  substance  resembling  Payen's 
" gelose."  In  1884,  Bauer  (169)  showed  that  agar-agar  yields  galac- 
tose; and  in  1905,  Konig  and  Bettels  (190)  gave  the  following  per- 
centage composition  of  Japanese  agar-agar  from  Gelidium: 

Per  cent.  Per  cent- 

Galactans   33       Ash   3.5 

Water   20       Pentosans   3. 1 

Protein   2.6     Crude  fiber   0.4 


xThe  term  agar-agar  is  applied  to  the  hot  water  extract  of  various  red  algae, 
mainly  species  of  Gelidium. 
2See  Pentosans,  p.  268. 


Nutrition  Investigations.  283 

Another  species  of  marine  algae  in  which  galactan  has  been  fully 
identified,  is  Chondrus  crispus  (Irish  moss).  This  is  also  a  red  alga. 
C.  Schmidt  (210)  first  examined  it,  in  1844;  he  demonstrated  that 
the  gelatinizing  substance  was  a  carbohydrate  and  yielded  sugar  on 
hydrolysis.  Fliickinger  and  Mayer  (178),  in  1868,  discovered  that 
the  water  extract  of  this  alga  yielded  consieterable  mucic  acid.  In 
1875,  Bente  (171)  obtained  levulinic  acid  from  the  products  of  its 
hydrolysis,  and  in  1876,  reported  that  it  yielded  a  non-crystallizing 
syrup  (172).  The  first  quantitative  analysis  was  made  by  Hadike, 
Bauer  and  Tollens  (185),  who  showed  that  the  water  extract  yielded 
mucic  acid  corresponding  to  about  25  per  cent  of  galactan.  Sebor 
(220),  in  1900,  found  in  the  products  of  hydrolysis,  glucose,  fructose 
and  a  small  quantity  of  pentose.  These  observations  were  verified 
by  Miither  (200)  in  1903,  who  further  identified  the  galactose  as  a 
(/-galactose.  From  the  large  yield  of  mucic  acid,  the  water  extract 
of  Chondrus  may  therefore  be  regarded  as  chiefly  galactan,  together 
with  some  dextran  and  levulan,  and  a  very  little  pentosan;  groups 
which,  according  to  Hadike,  Bauer  and  Tollens  (185),  may  be  partly 
or  entirely  bound  into  ester-like  compounds. 

Examples  of  galactans  occurring  in  combination,  or  close  associa- 
tion with  other  hemicelluloses  are  numerous.  Lupeose,  from  luzerne 
seeds,  originally  called  /3-galactan,  yields  50  per  cent  galactose  and  50 
per  cent  fructose  (214).  The  tuberous  roots  of  Stachys  tuberifera 
contain  a  soluble  crystallizable  carbohydrate  yielding  37  per  cent 
mucic  acid,  along  with  an  unidentified  sugar  (225).  Para-galactan 
(galacto-araban)  forms  a  large  proportion  of  the  reserve  material  of 
many  seeds.1  Rothenfusser  (204)  finds  that  the  mucilaginous  extract  of 
flaxseed  yields  equal  parts  of  pentosans  and  hexosans,  the  latter  being 
mainly  galactose.  Galactans  and  pentosans,  as  already  indicated,2 
occur  together  in  many  lichens  and  algae,  and  also  in  the  pectins.3 
Herissey  (187)  has  shown  that  the  "galactine"  of  Muntz  (199)  yields 
equal  parts  of  galactose  and  mannose.  Galacto-mannans  also  fre- 
quently occur  in  the  reserve  material  of  seeds,  as  in  those  of  the  date 
and  other  species  of  palm,  and  in  coffee  beans;  in  the  American  honey 


xCf.  Schulze  (215),  Schulze,  Steiger  and  Maxwell  (217),  Schulze  and  Castoro 
(218),  Castoro  (176),  and  Goret  (179).  Also  Schulze  and  Godet,  Zeitschrift  fur 
physiologische  Chemie,  V.  61,  p.  279,  for  a  very  complete  review  of  the  work  of 
Schulze  and  his  pupils. 

2See  Chemical  Nature  of  Lichens  and  Algae:-  Konig  and  Bettels  (8),  Escombe  (6), 
K.  Miiller  (11),  Ulander  (26). 

3Cf.  Pentosans,  p.  268. 


284 


Mary  Davies  Swartz, 


locust  (Gleditschia  triacanthus) ,  Goret  (179)  found  the  albumen  to 
yield  66-70  per  cent  galactose  and  22-23  per  cent  mannose;  he  has 
shown,  in  fact,  that  the  carbohydrate  reserve  of  almost  all  seeds  with 
horny  albumen  consists  largely  of  a  mixture  of  mannans  and  galac- 
tans.1 

GALACTANASES  IN  THE  VEGETABLE  KINGDOM. 

The  hydrolysis  of  the  paragalactan  of  lupine  seeds  during  germina- 
tion was  first  observed  by  Schulze  and  his  co-workers.  That  ordi- 
nary diastatic  enzymes  do  not  form  sugar  from  the  para-galactan  of 
Lupinus  hirsutus  was  demonstrated  by  Schulze  and  Castoro  (218). 
Ptyalin,  pancreatin,  malt  diastase  and"taka"  diastase,  will,  however, 
in  the  course  of  5  or  6  days'  digestion  at  35-40°  C.  render  this  carbo- 
hydrate soluble  in  water  to  the  following  extent: 

Per  cent.  Per  cent. 

Malt  diastase   38    Ptyalin   40 

Taka  diastase   35    Pancreatin   15 

,  Griiss  (184)  has  made  exhaustive  microchemical  investigations  upon 
the  germinating  date  endosperm,  in  which  he  has  been  able  to  observe 
the  solution  of  the  galactans  by  enzymes  developed  during  germina- 
tion. Bourquelot  and  Herissey  (174)  find  a  soluble  enzyme  hydrolyz- 
ing  galactan,2  produced  by  the  germinating  embryos  of  the  seeds  of 
the  carob,  Nux  vomica,  fenugrec  and  luzerne.  Shellenberg  (208), 
studying  the  action  of  moulds  on  hemicelluloses,  found  at  least  four 
different  ferments  showing  considerable  specificity  in  their  action; 
seeds  of  Lupinus  hirsutus  (containing  paragalactan)  were  attacked 
by  most  of  these  moulds  (Mucor  neglectus,  Mucor  piriforme,  Rhizopus 
nigricans,  Thamnidium  elegans,  Penicillium  glaucum).  Similarly, 
Herissey  (187)  found  galactose  produced  from  manno-galactans  by 
Aspergillus  niger  and  Aspergillus  fuscus;  Saiki  (205)  obtained  sugar 
from  Irish  moss  by  digesting  it  with  inulase  prepared  from  Aspergillus 
niger  and  Penicillium  glaucum;  and  with  "taka"  diastase  prepared 
from  another  mould,  Eurolium  oryzae. 

Little  is  known  of  the  action  of  bacteria  upon  galactans.  Gran 
(182)  found  sugar  produced  from  agar-agar  by  Bacillus  gelaticus, 
through  the  action  of  an  enzyme  which  he  calls  "  gelase."    Saiki  (105), 


^f.  Mannans,  p.  283;  for  a  further  discussion  of  the  occurrence  of  Galactans  see 
v.  Lippmann,  Chemie  der  Zuckerarten,  Vol.  I,  pp.  686-697. 
2Cf.  Mannans,  p.  284. 


Nutrition  Investigations. 


285 


in  experiments  with  B.  coli  communis,  on  culture  media  containing 
different  kinds  of  comminuted  seaweed,  found  a  slight  gas  production 
in  one  culture,  in  media  with  agar-agar  and  Irish  moss. 

GALACTANASES  IN  THE  ANIMAL  KINGDOM. 

The  only  discovered  instance  of  a  galactanase  in  lower  animals  is 
cited  by  Bierry  and  Giaja  (173),  who  found  that  the  hepato-pan- 
creatic  juice  of  Helix  pomatia  produced  galactose  from  extracts  of 
carob  seeds  (Ceratonia  siliqua) ;  later  experiments  upon  agar-agar, 
with  extracts  from  a  number  of  crustaceans  (Astacus  fluviatilis 
Rondel.,  Homarus  vulgaris  Bel.,  Maja  squinado  Rondel.,  Carcinus 
moenas  L.,  and  Platycarcinus  pagarus  L.)  were  entirely  negative;  the 
galactans  of  luzerne  and  fenugrec  were  attacked  with  difficulty  by  the 
extract  from  Astacus.  Strauss  (221)  could  find  no  enzyme  attack- 
ing agar-agar,  in  the  larvae  and  puppae  of  various  species  of  Lepidop- 
tera  and  Diptera. 

No  galactanases  have  been  found  in  higher  animals.  Bierry  and 
Giaja  (173),  using  extracts  of  luzerne  seeds,  got  negative  results 
with  digestive  juices  of  dogs  and  rabbits ,  and  Sawamura  (207)  ob- 
tained similar  results  with  extracts  of  different  sections  of  the  alimen- 
tary canal  of  swine  and  horses.  Saiki  (205)  found  saliva,  pancreatic, 
and  intestinal  juices  unable  to  hydrolyze  Irish  moss. 

DIGESTION  AND  UTILIZATION  OF  GALACTAN  BY  ANIMALS  AND  MAN. 

The  first  study  of  the  digestibility  of  galactans  in  higher  animals 
was  made  in  1903,  by  Lindsey  (191).  Alsike  clover-seed,  containing 
8  per  cent  galactan,  was  fed  in  connection  with  hay,  the  digestibility 
of  which  had  been  previously  determined;  from  analyses  of  food  and 
faeces,  the  galactan  in  the  hay  (1.72  per  cent)  was  found  to  be  75  per 
cent  digestible,  and  that  in  the  clover  95.78  per  cent  digestible. 
Saiki  (205)  fed  agar-agar  and  Irish  moss  to  dogs  and  recovered  a  large 
part  in  the  faeces,  as  shown  by  the  increased  amount  of  carbohydrate 
excreted.  Lohrisch  (194)  fed  dogs  and  rabbits  agar-agar  in  its  usual 
form,  and  also  "  soluble-agar "  prepared  from  ordinary  agar  by  Dr. 
Karl  Dieterich  of  Dresden,  Director  of  the  Helfenberg  Chemical  Fac- 
tory. This  product  seems  to  be  partially  hydrolyzed  in  its  prepara- 
tion, since  it  is  not  only  readily  soluble  in  water,  but  has  slight  reduc- 
ing action;  it  yields  on  boiling  with  Fehling's  solution,  3.5^.1  per 
cent  sugar,  and  if  a  watery  solution  is  allowed  to  stand  18  hours  at 


286 


Mary  Davies  Swartz, 


37°  C,  it  is  further  hydrolyzed  and  yields  then  16.9-20.4  per  cent  sugar. 
The  results  of  Lohrisch's  experiments  appear  in  the  folowing  table: 


ANIMAL. 

FOOD. 

HEMICELLULOSE 
EQUIVALENT  OF  AGAR 
FED. 

HEMICELL- 
ULOSE 
EXCRETED. 

HEMICELL- 
ULOSE 
DIGESTED. 

Per  cent. 

Rabbit  I  

Ordinary  agar 

18.77  =  14.48 

7.1 

50.9 

Rabbit  II  

Ordinary  agar 

11.8    =  9.11 

4.71 

48.3 

Rabbit  III  

Soluble  agar 

95.9    =  65.02 

14.2 

78.1 

(given  in  9  days) 

Dog  

Same  as  III 

53.0    =  35.9 

11.7 

67.3 

Lohrisch  (194)  has  also  studied  the  utilization  of  agar-agar  in  starv- 
ing herbi'vora.  In  two  experiments,  rabbits  starved  for  two  days 
were  fed  ordinary  agar  as  long  as  they  would  eat  it,  other  animals  of 
the  same  weight  being  kept  in  starvation  as  controls;  in  a  third  expe- 
riment, " soluble  agar"  was  fed.  Urine  and  faeces  were  collected  and 
analyzed.  Of  the  ordinary  agar,  about  50  per  cent  was  excreted  in 
the  faeces;  of  " soluble  agar,"  about  25  per  cent.  No  positive  evidence 
of  any  change  in  nitrogen  excretion  attributable  to  the  agar  fed,  can 
be  drawn  from  the  protocols.  One  animal  died  through  accident, 
another  survived  its  control  but  one  day,  and  the  third,  in  spite  of  its 
apparently  good  digestion  of  the  "soluble  agar,"  died  a  week  before 
its  control. 

In  the  case  of  rabbits  made  diabetic  with  phlorhizin  and  then  fed 
20-40  grams  of  both  ordinary  and  soluble  agar,  Lohrisch  (194)  found 
that  the  D :  N  ratio  remained  fairly  constant  throughout  each  experi- 
ment, showing  no  marked  increase  in  sugar  excretion.  We  see,  there- 
fore, no  grounds  for  assuming  that  agar-agar  (galactan)  forms  glycogen 
in  rabbits. 

The  first  studies  on  the  utilization  of  galactan  by  man  were  made 
by  Saiki  (205)  (1906).  In  feeding  experiments  in  which  various  car- 
bohydrates were  at  different  times  added  to  a  uniform  diet,  consisting 
of  513  grams  beefsteak,  500-600  grams  bread,  40  grams  sugar,  31 
grams  butter,  2  eggs  and  2  apples  —  a  diet  on  which  over  98  per  cent 
of  the  carbohydrates  were  digested,  he  obtained  the  following  results: 


Nutrition  Investigations . 


287 


NO. 

SUBSTANCE   ADDED   TO  DIET. 

EQUIVALENT  OF 
SUBSTANCE  IN 
DEXTROSE. 

CARBOIIYDRATES 
IN  FAECES  CAL- 
CULATED AS 
DEXTROSE. 

HEMICELLULOSE 
DIGESTED. 

Grams. 

Grams. 

Per  cent. 

1 

10 

9.2 

8 

2 

24  grams  agar  

12 

8.8 

27 

3 

40  grams  wakame  

4.7 

3.4 

28 

4 

11.4 

2.5 

78 

Lohrisch  has  also  studied  the  digestibility  of  "soluble  agar"  in 
man.  Sometimes  it  is  not  well  borne,  especially  if  given  in  quanti- 
ties over  50-60  grams  per  day  and  causes  gas  formation,  diarrhoea, 
and  other  intestinal  disturbances;  in  other  cases,  large  amounts  (100 
grams  per  day)  cause  no  unpleasant  symptoms  whatever.  The  agar 
was  dissolved  in  some  beverage,  and  the  diet  was  otherwise  carbohy- 
drate-free.   Some  of  the  results  are  shown  in  the  following  table  (194) : 


NO. 

DURATION  OF 
EXPERIMENT. 

AMOUNT 

DIGESTED. 

HEMICELL- 
ULOSE 
DIGESTED. 

HEMICELL- 
ULOSE 
DIGESTED. 

As  Air  Dry 
Soluble  Agar. 

As  Hemicel- 
lulose. 

HEMICELLULOSE 
EXCRETED. 

Grams. 

Grams. 

Grams. 

Grams. 

Per  cent. 

1 

1  day 

100 

61.9 

46.06 

15.84 

25.6 

2 

1  day 

100 

61.9 

39.1 

22.8 

36.8 

3 

3  days 

235 

145.4 

90.5 

54.9 

37.7 

4 

3  days 

240 

148.5 

40.8 

107.7 

72.5 

5 

1  day 

100 

61.9 

25.4 

36.5 

58.9 

6 

1  day 

110 

67.8 

23.4 

44.4 

65.5 

No.  4  was  a  case  of  chronic  constipation;  the  high  percentage  of  hemi- 
cellulose  digested  is  in  accordance  with  the  observations  of  Lohrisch 
(193)  and  Pletnew  (203),  on  the  extraordinarily  good  utilization  of  all 
foodstuffs  in  chronic  constipation.  Two  of  these  experiments  were  on 
diabetics,  and  showed  that  the  18.36  grams  of  "soluble  agar"  ab- 
sorbed per  day  caused  no  increase  of  sugar  in  the  urine,  and  had  no 
noticeable  effect  on  nitrogen  metabolism. 

From  these  experiments,  we  see  that  ordinary  agar  is  digestible  to  a 
very  small  extent,  and  that  even  when  changed  to  an  easily  hydro- 
lyzed  form,  it  is  only  digested  to  about  50  per  cent.  Is  the  part 
digested  absorbed  and  utilized  as  galactose?    The  recent  exhaustive 


288 


Mary  Davies  Swartz, 


discussion  of  the  behavior  of  galactose  in  the  animal  body  by  Brasch 
(175)  renders  any  details  on  the  utilization  of  this  sugar  unnecessary. 
Hofmeister  (188)  showed  that  of  all  sugars  it  is  most  readily  excreted. 
That  galactose  can  form  glycogen  in  dogs  and  rabbits,  has  been  shown 
by  Weinland  (226),  Kausch  and  Socin  (189),  Cremer  (177),  Voit  (223), 
Brasch  (175),  and  others.1  Brasch  (175)  has  shown  that  the  assimila- 
tion limits  for  galactose  lie,  for  normal  man,  between  30  and  40  grams, 
while  for  dextrose  they  lie  between  100  and  150  grams.  Voit  (224), 
Sandmeyer  (206), Bauer  (170),  and  others  have  shown  that  galactose, 
even  in  small  amounts  increases  the  sugar  excretion  in  diabetes.  It 
would  seem,  therefore,  that  if  soluble  agar  were  absorbed  as  sugar,  it 
would  increase  the  sugar  output  in  the  urine.  To  throw  some  light 
on  this  problem  Lohrisch  (194)  has  conducted  three  respiration  ex- 
periments on  men  after  ingestion  of  100-110  grams  of  soluble  agar,  of 
which,  on  the  average,  about  63  per  cent  was  absorbed.  The  changes 
in  the  respiratory  quotient  are  shown  in  the  following  table: 


Respiratory  Quotient. 


NO. 

IN  FASTING. 

NUMBER  OF  HOURS  AFTER  INGESTION  OF  SOLUBLE  AGAR. 

1 

2 

3 

4 

5 

6 

7 

I 

II 
III 

0.768 

0.786 
0.739 

0.768 

0.766 

0.835 
0.794 
0.815 

0.860 
0.825 
0,800 

0.770 
0.767 
0.774 

0.735 

0.724 
0.714 

NO. 

IN  FASTING. 

NUMBER  OF  HOURS  AFTER  INGESTION  OF  SOLUBLE  AGAR. 

8 

9 

10 

n 

12 

13 

I 

II 

III 

0.768 
0.786 
0.739 

0.693 

0.730 
0.703 

0.618 

0.669 

The  distinct  rise  in  the  respiratory  quotient  in  the  fourth  hour 
(beginning  in  the  third  hour  in  Experiment  I)  would  indicate  that  car- 
bohydrate was  being  oxidized,  which  in  this  case  must  come  from  the 
agar.  The  low  value  in  the  later  hours  seems  due  to  the  oxidation  of 
fatty  acids;2  that  such  acids  may  be  formed  from  soluble  agar  by 
bacteria,  appears  probable  also  from  the  intestinal  fermentation  pro- 


*Cf.  Magnus-Levy,  Verwerthbarkeit  der  Galactose  in  normalen  Organismus :  Op- 
penheimer's  Handbuch  der  Biochemie  der  Menschen  und  der  Tiere,  Vol.  IV,  p.  379. 
2Cf .  respiration  experiments  described  under  Cellulose. 


Nutrition  Investigations. 


289 


duced  when  large  amounts  of  this  preparation  are  taken.  A  slight 
increase  in  acetone  output,  shown  in  the  metabolism  experiments 
with  diabetics,  points  to  the  same  conclusion.  Perhaps,  as  Lohrisch 
suggests,  the  very  slow  digestion  of  the  carbohydrate,  may  enable  the 
organism  to  utilize  the  galactose  formed,  and  account  for  its  non-ex- 
cretion, but  this  requires  further  demonstration. 

According  to  these  experiments  by  Lohrisch,  cellulose  and  the  solu- 
ble galactan  show  little  difference  in  their  physiological  behavior. 
Both  can  be  digested  to  about  50  per  cent.  Ordinary  agar,  as  Saiki's 
experiments  show,  is  largely  recovered  in  the  faeces;  in  fact, a  thera- 
peutic practice  which  has  been  recently  established  is  based  upon  the 
recognized  indigestibility  of  agar,  namely,  its  employment  as  a  remedy 
in  cases  of  chronic  constipation.  It  is  especially  valuable,  as  Mendel 
(196)  points  out,  in  those  cases  where  the  difficulty  is  due  to  an  ex- 
tremely complete  digestion  and  absorption  of  all  foodstuffs  from  the 
alimentary  tract,  which  causes  the  formation  of  dry,  hard  faecal 
masses  (scyballa)  difficult  to  evacuate.  The  agar,  remaining  undigested 
and  retaining  a  high  percentage  of  water,  gives  bulk  and  softness  to 
the  faeces,  and  facilitates  their  daily  elimination.  Being  resistant 
towards  bacterial  action,  it  causes  neither  gas  formation  nor  produc- 
tion of  harmful  decomposition  products.  According  to  A.  Schmidt 
(209),  it  can  be  advantageously  taken  in  quantities  up  to  25  grams  per 
day,  part  with  the  breakfast  cereal,  and  part  with  sauce  or  cream,  at 
another  meal.  In  view  of  such  facts  as  these,  we  are  hardly  prepared 
to  agree  with  Lohrisch,  that '  Cellulose  and  Hemicelluloses  are  readily 
digested. ' 

Occurrence  and  Nature  of  Mannans. 

As  widely  diversified  in  origin  and  character  as  the  galactans,  and 
very  intimately  associated  with  them  are  the  Mannans.  They  show 
all  possible  degrees  of  solubility,  from  the  readily  soluble  mucilage 
found  in  certain  legumes,  to  the  completely  insoluble  "reserve-cellu- 
lose," which  forms  the  horny  albumen  in  such  seeds  as  the  date,  and 
which  was  long  confused  with  true  cellulose. 

A  few  examples  will  serve  to  show  the  diverse  places  in  which  man- 
nans may  be  found.  They  occur  in  yeast:1  (258)  in  algae,  as  Por- 
phyra  laciniata;  (278)  in  moulds,  as  Penicillium  glaucum;  (285)  in  the 
leaves  and  roots  of  the  Japanese  plant,  Conophallus  konjaku  (280) ; 
in  the  bark  and  wood  of  many  American  trees  (272). 


*For  further  discussion  see  v.Lippmann,  Chemie  der  Zuckerarten,  Vol.1,  pp.  641- 
649,  and  Czapek,  Biochemie  der  Pflanzen,  pp.  325-329. 


290 


Mary  Barnes  Swartz, 


The  most  extensive  study  has  been  given  to  the  mannans  of  various 
seeds,  in  which,  as  already  shown,1  mannans  and  galactans  seem  al- 
most invariably  to  occur  together.  The  seeds  of  the  carob  tree  (Ce- 
ratonia  siliqua)  contain  a  hemicellulose  originally  called  "caruban" 
by  Effront  (241)  (1897),  but  shown  by  van  Ekenstein  (282)  to  yield 
mannose,  and  by  Bourquelot  and  Herissey  (232)  (1899),  ^-galactose. 
The  first  elaborate  studies  of  "reserve-cellulose"  were  made  by  Reiss 
(264),  who  showed  that  the  horny  albumen  of  the  seeds  of  Phytelepas 
macrocarpa,  Phoenix  dactylifera  and  other  species  of  palm,  Allium 
cepa,  Asparagus  officinalis,  Iris  pseudacorus,  Strychnos  nux  vomica 
and  Caffea  arabica,  differed  chemically  from  true  cellulose  in  their 
color  reactions,  in  the  ease  with  which  they  can  be  hydrolyzed,  and  in 
yielding,  instead  of  dextrose,  a  sugar  which  he  called  "seminose," 
but  which  proved  to  be  identical  with  Fischer  and  Hirschberger's  (242) 
previously  described  mannose. 

Mannan  also  occurs  richly  in  the  tubers  of  the  many  species  of  Or- 
chis and  Eulophia  which  are  the  source  of  commercial  salep.  On  ex- 
traction with  water,  they  yield  a  mucilaginous  extract  which  was 
first  studied  by  C.  Schmidt  (270)  in  1844,  and  called  by  him  "salep- 
bassorin";  on  hydrolysis  with  dilute  sulphuric  acid  he  obtained,  be- 
s'des  some  gummy  substance  and  cellulose,  a  fermentable  sugar 
which  he  thought  to  be  dextrose.  Mulder  (259)  considered  the  salep 
mucilage  a  mixture  of  starch  and  gum  or  pectin  acids,  while  Franck 
(243)  thought  it  a  modification  of  cellulose,  and  Girand  (248)  a  trans- 
formation of  a  starchy  substance  into  a  variety  of  dextrin  swelling  in 
water.  Pohl  (263)  by  precipitation  with  neutral  salts,  distinguished 
an  "a-Schleim"  and  a  "jS-Schleim. "  According  to  Thamm  (276),  who 
has  made  the  most  recent  investigations,  "a-Schleim"  does  not  occur 
in  German  salep.  Tollens  and  Gans  (277)  showed  that  on  hydrolysis, 
besides  dextrose,  mannose  or,  as  they  called  it,  "  isomanitose "  was 
formed,  but  this  was  shown  by  Fischer  and  Hirschberger  (242)  to  be 
identical  with  d-mannose.  Thamm  (276)  and  Hilger  (254)  have  shown 
conclusively,  that  the  starch-free  water  extract  contains  an  anhydride 
of  mannose  only. 

A  very  resistant  type  of  mannan  occurring  in  some  plants,  has  been 
designated  as  manno-cellulose  by  Schulze  (273).  Bertrand  (227) 
finds  it  taking  the  place  of  xylan  in  the  woody  tissues  of  gymnosperms. 


xCf .  Schulze  and  his  coworkers,  and  Goret,  under  Galactans.  Also  Schulze  and 
Godet,  Zeitschrift  fur  physiologische  Chemie,  V.  61,  p.  279,  for  a  very  complete 
review. 


Nutrition  I  n  vest  i  gat  ions . 


291 


MANNANASES  IN  THE  VEGETABLE  KINGDOM. 

There  is  very  little  literature  concerning  the  action  of  bacteria  upon 
mannans.  Sawamura  (267)  observed  that  extracts  of  Hydrangea  pa- 
niculata,  used  in  the  manufacture  of  Japanese  paper,  which  contain 
mannan  (along  with  galactan  and  araban) ,  became  liquefied  on  stand- 
ing. In  bacteriological  studies  with  extracts  of  this  plant,  and  of 
roots  of  Cono phallus  konjaku,  he  found  that  only  B.  mes enter icus  ml- 
gatus  dissolved  these  mannans.  The  action  was  greatly  facilitated, 
and  sugar  formation  increased  if  a  certain  wild  yeast,  in  itself  inactive, 
were  present.  Traces  of  a  similar  enzyme  seem  to  occur  in  B.  prodi- 
giosus. 

In  his  studies  of  the  action  of  moulds  on  hemicelluloses,  Schellen- 
berg  (269)  found  that  the  seeds  of  Ruscus  aculeata,  which  yield  almost 
exclusively  mannose  (237-240),  were  attacked  only  by  Penicillium 
glaucum.  Herissey  (253),  using  pure  cultures  and  water  extracts  of 
cultures  of  Aspergillus  niger  (grown  on  media  rich  in  mannose  and  ga- 
lactose to  incite  the  development  of  mannanase  and  galactanase),  with 
suitable  antiseptics  and  controls,  obtained  mannose  —  and  galactose 
—  from  seeds  of  Ceratonia  siliqua  and  Gleditschia  triacanthus,  and  an 
abundant  yield  of  mannose  from  salep;  similar  results  were  obtained 
with  Aspergillus  fuscus. 

As  early  as  1862,  Sachs  (266)  observed  the  change  of  the  thickened 
cell-walls  of  the  date  endosperm  into  sugar  during  germination.  The 
cytases  producing  this  change  in  'reserve-cellulose'  were  later  care- 
fully investigated  by  Reiss  (264),  Brown  and  Morris  (230),  Newcombe 
(261),  Gniss  (251),  and  others.  Still  more  recently,  Bourquelot  and 
Herissey  have  made  many  studies  on  the  specific  characteristics  of 
these  plant  enzymes.  An  exhaustive  review  of  the  literature  on  man- 
nans and  the  action  of  enzymes  upon  them  has  been  published  by 
Herissey  (253),  consequently  this  subject  will  only  be  reviewed  very 
briefly  here. 

Griiss  (251)  has  demonstrated  that  the  solution  of  the  date  embryo 
(Phoenix  dactylifera)  is  due  to  a  ferment,  the  product  of  whose  activ- 
ity is  galactan  and  mannose.  Effront  (241)  (in  1897)  attributed  the 
solution  of  the  albumen  of  carob  seeds  (called  by  him  caruban)  to  a 
"caroubinase,"  but  thought  that  the  product  of  its  activity  was  not 
identical  with  the  products  of  hydrolysis;  in  1899,  however,  Bourque- 
lot and  Herissey  (233)  showed  the  possibility  of  obtaining  mannose 
by  the  action  of  a  soluble  ferment  derived  from  these  seeds,  which 
they  called  "seminase."    Shortly  afterwards,  a  similar  enzyme  was 


292 


Mary  Davies  Swartz, 


isolated  by  them  from  the  seeds  of  Phoenix  canariensis.  Herissey 
(253)  has  been  able  to  show  that  seeds  of  such  legumes  as  luzerne, 
fenugrec,  and  common  genet  have,  at  least  at  the  time  of  germination, 
ferments  capable  of  transforming  mannans — and  galactans — into 
their  corresponding  sugars.  Experiments  in  vitro  show  that  they  are 
not  limited  to  action  upon  the  seeds  by  whose  embryos  they  are  pro- 
duced, but  act  on  the  reserve-cellulose  of  seeds  from  very  distinct 
groups  of  plants.  However,  the  luzerne  ferment  does  not  digest  all 
mannans  and  galactans;  it  will  hydrolyze  the  mannans  of  the  tubers 
of  the  Orchis  family  (and  commercial  salep  prepared  from  them),  but 
not  those  of  the  albumen  of  palm  seeds. 

Griiss  (251)  has  also  shown  that  the  enzyme  of  the  date  endosperm 
hydrolyzes  starch,  although  this  does  not  occur  in  the  date  seed,  and 
that  malt  diastase  works  on  a-mannan  (the  soluble  mannan  of  date 
seeds,  according  to  Griiss)  which  does  not  occur  in  the  barley  endo- 
sperm. Griiss  considers  diastatic  enzymes  a  group  working  not  only 
on  starch,  but  also  on  hemicelluloses.  Herissey  thinks  that  diastase 
and  seminase  are  found  together  in  varying  proportions  in  barley, 
legumes,  carob  seeds,  etc.,  and  that  neither  is  a  simple  ferment,  but  a 
" superposition  de  ferments,"  and  defines  "seminase"  as  a  "ferment 
or  group  of  soluble  ferments,  causing  the  transformation  of  the  car- 
bohydrates of  horny  albumens  of  the  seeds  of  Leguminosae  into  as- 
similable sugars."  Gatin  (247)  has  made  further  researches  upon  the 
nature  of  seminase,  and  states  that  during  the  germination  of  certain 
seeds  whose  reserve  is  in  the  form  of  mannan,  the  presence  of  mannose 
is  exceptional,  but  dextrose  occurs  in  abundance.  This  phenomenon 
he  attributes  to  a  "manno-isomerase,"  which  transforms  the  mannose, 
as  fast  as  formed  by  the  seminase,  into  dextrose.  Experiments  in 
vitro  seem  to  indicate  that  this  is  a  soluble  ferment. 

MANNANASES  IN  THE  ANIMAL  KINGDOM. 

There  are  only  a  few  instances  on  record  of  mansases  occurring  in 
lower  animals.  Bierry  and  Giaja  (228,  229)  found  that  the  hepato- 
pancreatic  juice  of  Helix  pomatia  was  capable  of  producing  mannose 
from  extracts  of  carob  seeds  and  salep;  that  of  Astacus  fluviatilis,  Ho- 
marus  vulgaris,  and  Maja  squinado,  from  the  ivory  nut  (Phytelepas  ma- 
crocarpa) ,  the  two  latter  hydrolyzing  it  at  ordinary  room  temperature. 
On  the  other  hand,  the  mannans  of  fenugrec  and  luzerne  were  hydro- 
lyzed  with  difficulty,  or  not  at  all,  by  very  pure  gastro-intestinal 
juice.    No  mannanase  was  found  by  Strauss  (275)  in  the  larvae  and 


Nutrit  ion  Investigations 


293 


puppae  of  Lepidoptera  and  Diptera.  Similar  negative  results  have 
been  obtained  with  the  digestive  enzymes  of  higher  animals.  Kino- 
shita  (257)  found  that  emulsin  and  invertin  did  not  hydrolyze  the  man- 
nans  of  Conophallus  konjaku  and  Gatin  (245,  246)  tried  the  blood  of 
rabbits,  chicken  serum,  the  pancreatic  juice  of  dogs,  the  macerated 
intestines  and  pancreas  of  chickens  and  cattle,  upon  salep  and  carob 
seeds  with  negative  results;  on  the  other  hand,  Sawamura  (268)  re- 
ports a  mannanase  in  the  extracts  from  different  sections  of  the  ali- 
mentary tract  of  swine  and  horses. 

DIGESTION  AND  UTILIZATION  BY  ANIMALS  AND  MAN. 

There  are  also  very  few  records  in  the  literature  of  feeding  experi- 
ments with  mannans.  In  a  paper  in  the  Zeitschrift  fur  Biologie,  Voit 
(283)  in  18741  described  one  by  Hauber,  who  fed  a  medium  sized  dog 
390  grams  of  dry  salep  powder  in  the  course  of  eight  days.  The  faeces 
of  the  feeding  period  were  roughly  marked  off,  and  Hauber  reported 
no  unchanged  salep  present  in  them,  because  there  was  no  swelling 
in  water  as  with  the  original  powder.  Calculations  based  on  the  yield 
of  sugar  from  the  faeces  on  hydrolysis  showed  that  at  least  50  per  cent 
of  the  salep  was  absorbed.  This  seems  to  have  been  a  very  crude  ex- 
periment, and  cannot  be  considered  of  convincing  value. 

In  1879,  Weiske  (284)  fed  carob-beans  {Ceralonia  siliqua)  to  sheep, 
along  with  meadow  hay,  and  compared  the  nutritive  value  of  this  ra- 
tion with  one  in  which  the  carob-beans  (210  grams)  were  replaced  by 
an  equivalent  weight  of  starch,  sugar  and  protein  (from  crushed  peas). 
The  coefficients  of  digestibility  and  nitrogen  balance  were  so  nearly 
the  same  on  the  two  rations,  that  Weiske  pronounced  "Johannis- 
brod"  (carob  beans)  an  acceptable  and  digestible  feed  for  sheep. 

In  1890,  Schuster  and  Liebscher  (274)  tried  feeding  the  sawdust  of 
ivory  nut  (Phytelepas  macrocarpa)  to  sheep,  having  previously  found 
that  it  had  a  favorable  effect  on  cattle.  Merino  sheep  gained  consider- 
able fat  when  fed  oat  straw  and  vetch  fodder,  plus  ivory  nut  sawdust 
furnishing  50  per  cent  of  the  digestible  carbohydrates.  The  ration, 
exclusive  of  the  ivory  nut,  did  not  yield  enough  energy  for  such  a  re- 
sult to  be  possible,  hence  the  latter  must  have  been  utilized.  The 
coefficient  of  digestibility,  both  for  the  nitrogen-free  extract  and  crude 
fiber  of  this  material,  was  at  the  same  time  shown  by  Niebling  (262) 
to  be  82  per  cent  for  sheep. 


xThis  paper  reviews  the  early  literature  on  gums. 


294 


Mary  Davies  Swartz, 


From  these  experiments,  mannan  would  seem  to  be  well  utilized  by 
herbivora.  The  only  experimental  data  regarding  the  nutritive  value 
of  mannans  to  man,  are  cited  by  Oshima  (15)  from  work  by  Kano  and 
Iishima  (255),  who  found  the  coefficient  of  digestibility  of  konjaku  82 
per  cent  (prepared from Conophallus  konjaku).  Further  investigations 
seem  highly  desirable,  in  view  of  the  fact  that  in  certain  regions  food 
stuffs  like  salep  and  konjaku,  consisting  of  almost  pure  mannan,  are 
among  the  chief  articles  of  the  poor  man's  diet.  It  is  also  a  question 
whether  the  nutritive  value  of  bark,  especially  of  coniferous  trees,  is 
due  to  mannan  present.  According  to  Dillingham  (239)  the  quantity 
of  mannan  present  does  not  justify  such  an  assumption,  aside  from 
the  question  of  its  digestibility. 

We  have  finally  to  inquire  whether  mannan  can  be  hydrolyzed 
within  the  organism,  and  if  so,  whether  the  mannose  produced  can  be 
retained  and  form  glycogen.  From  the  literature  on  the  subject,  it 
appears  that  mannose  is  well  utilized  by  rabbits,  dogs  and  men.  Ac- 
cording to  Neuberg  and  Mayer  (260),  the  d-form  is  better  utilized 
than  the  /-  or  i-form.  Mannose  is  readily  converted  to  dextrose  in 
the  organism;  thus  Neuberg  and  Mayer  found  that  a  rabbit,  receiv- 
ing 10  grams  of  /-mannose  per  os,  excreted  1  gram  /-mannose  and  4-5 
grams  /-glucose;  10  grams  of  ^-mannose  given  rabbits  per  os,  or  sub- 
cutaneously,  were  almost  completely  oxidized.  Rabbits  fed  30  grams 
d-mannose  by  Cremer  (238)  excreted  3-4  grams  in  the  urine,  and  dogs 
given  20  grams  by  Rosenfeld  (265),  excreted  over  4  grams.  This  is 
somewhat  more  than  would  be  excreted  on  giving  equally  large  quan- 
tities of  dextrose  or  levulose.  Cremer  (238)  found  no  sugar  in  the 
urine  of  a  man  after  feeding  3-12  grams  of  mannose. 

That  mannose  can  act  as  a  glycogen  former  in  rabbits,  has  been 
demonstrated  by  Cremer  (238)  and  also  by  Rosenfeld  (265).  Neu- 
berg and  Mayer  (260)  found  only  a  small  amount  of  glycogen  in  the 
livers  of  starving  rabbits  after  feeding  /-mannose,  but  even  this  form 
is  utilized  to  some  extent.  There  is  good  reason  for  assuming,  there- 
fore, that  if  mannans  can  be  converted  into  mannose  in  the  process 
of  digestion,  they  may  be  considered  as  true  nutrients  for  the  organ- 
ism, the  mannose  being  to  a  high  degree  capable  of  absorption  and  con- 
version into  glycogen. 


Nutrition  Invest iga Hons. 


295 


Occurrence  and  Nature  of  Levulans. 

A  number  of  polysaccharide  carbohydrates  yielding  levulose  on 
inversion  have  been  described.  They  are  all  levo-rotatory,  more  or 
less  soluble  in  cold  water  and  insoluble  in  alcohol,  and  easily  hydro- 
lyzed  by  dilute  acid,  but  have  not  been  investigated  sufficiently  to 
permit  any  conclusion  to  be  drawn  respecting  their  relation  to  one 
another.  The  most  important  of  these  substances  and  their  sources 
are  shown  in  the  following  table:* 


NAME. 

SOURCE. 

INVESTIGATOR. 

Inulin 

Tubers  of  dahlia,  artichoke,  Jerusalem 
artichoke,  elecampane;  bulbs  of  onion, 
garlic,  narcissus,  hyacinth,  and  tube- 
rose; flowers,  seed,  etc.,  of  various 
compositae 

Tanret  (321) 
Chevastelon  (291) 

Pseudo-inulin 
Inulenin 
Helianthin 
Synanthrin 

Tubers  of  dahlia,  artichoke,  Jerusalem 
artichoke,  elecampane;  bulbs  of  onion, 
garlic,  narcissus,  hyacinth,  and  tube- 
rose; flowers,  seed,  etc.,  of  various 
compositae 

Tanret  (321,  322) 

Levulin 

Tubers  of  Helianthus  tuberosus  (Jeru- 
salem artichoke) 

Reidemeister  (314) 
and  others 

Phlein 

Rootstalks  of  Phleum  praetense  (Tim- 
othy) 

Ekstrand   and  Jo- 
hanson  (296) 

Cerosin 

Unripe  grains 

Tanret  (320) 

Graminin 

Rootstalks  of  various  grasses,  e.g., 
Trisetum  alpestre 

Ekstrand  and  Johan- 

son  (296) 
Harlay  (301) 

Triticin 

Dracaena  australis  and  rubra,  Triti- 
cum  repens  (couch  grass) 

Reidemeister  (314) 

Sinistrin 

Bulbs  of  Scilla  Maritima  (Sea  onions 
or  squills) 

Schmiedeberg  (318) 
Reidemeister  (314) 

Levulan 

Molasses  in  beet-sugar  industry 

v.  Lippmann  (309) 

*  Cf.  v.  Lippmann,  Chemie  der  Zuckerarten,  Vol.  I,  pp.  795-807. 


296 


Mary  Davies  Swartz, 


The  best  known  member  of  this  group  is  inulin,1  closely  associated 
with  which  are  the  four  levulans  described  by  Tanret;  these  seem  to 
be  intermediate  products  between  inulin  and  levuiose,  all  having 
greater  solubility  than  inulin,  but  less  levo-rotatory  power.  The 
other  carbohydrates  mentioned  are  also  more  soluble  than  inulin, 
but  have  higher  specific  rotation. 


LEVULANASES  IN  THE  VEGETABLE  KINGDOM. 


Comparatively  few  studies  have  been  made  upon  the  action  of 
enzymes  on  the  levulans,  and  these  have  been  for  the  most  part  lim- 
ited to  inulin.  Certain  micro-organisms  as  B.  Coli  communis  (295), 
Clostridium  pastorianum  (328),  and  several  Schizomycetes,  decom- 
pose inulin,  but  without  any  production  of  sugar.  Yeast,  according 
to  Tanret  (321)  does  not  ordinarily  ferment  it,  but  Lindner  (308) 
asserts  that  certain  forms  of  top  yeast  change  it  readily.  Levulin 
is  fermented  by  yeast,  according  to  Levy  (307),  and  triticin,  in  the 
course  of  four  or  five  days,  according  to  Reidmeister  (314);  but  it 
seems  probable  that  the  first  changes  are  due  to  gradual  hydrolysis 
on  standing  in  water,  or  to  other  organisms. 

The  effect  of  vegetable  enzymes  on  these  carbohydrates,  as  far  as 
they  have  been  studied,  is  shown  in  the  following  table: 


NAME  OF  LEVULAN. 


Inulin 
Levulin .  . 
Graminin . 

Triticin.  . 
Sinistrin  . 


INVERTIN 
OF  YEAST. 


-  (8) 
+  (D 


(2) 


MALT  DIASTASE. 


-(3) 

-(4) 

+  (5) 
-(6) 


TAKA  DIASTASE. 


(3) 


INULASE  OF 
ASPERGILLUS. 


+  (7) 

+  very 
slowly  (4) 


(1)  Levy  (307) 

(2)  Reidemeister  (314) 

(3)  Chittenden  (292) 

(4)  Harlay  (301) 


(5)  Reidemeister  (314) 

(6)  Schmiedeberg  (318) 

(7)  Dean  (293)  and  others 

(8)  Komanos  (303) 


Discovery  of  the  best  known  ferment  for  any  levulan  is  due  to 
Green  (300)  who,  in  1888,  extracted  such  an  enzyme  from  the  tubers 
of  the  Jerusalem  artichoke  (Helianthus  tuberosus),  and  named  it  "in- 


xFor  description  and  early  literature  see  Kiliani  (302)  and  Dean  (294). 


Nutrition  I  nvestigations. 


297 


ulase. "  Subsequently,  Bourquelot  (289)  found  inulase  in  Asper- 
gillus niger  and  Penicillium  glaucum;  and  Chevastelon  (291)  showed 
that  this  enzyme  would  hydrolyze  the  inulin  of  the  monoctyledons. 
Dean  (293)  has  studied  the  properties  of  inulase  exhaustively,  and 
shown  that  in  Aspergillus  and  Penicillium  it  exists  only  as  an  endo- 
enzyme.  Went  (327)  has  found  inulase  also  in  Monilia  sitophila  and 
other  Amylomyces. 

LEVULANASES  IN  ANIMALS. 

The  first  instance  of  an  inulase  in  an  animal  organism  has  been 
cited  by  Strauss  (319).  In  1908,  he  reported  studies  on  the  enzymes 
of  seven  species  of  Lepidoptera  and  Diptera,  during  their  various 
stages  of  development  (Euproctis  chrysorrhea,  Ocneria  disparata,  Bom- 
byneustria,  Bombyx  mori,  Galleria  melonella,  Hyponomenta,  Calliophera 
vomitoria),  but  found  inulase  present  only  in  the  eating  larvae  of 
Bombyx  mori  and  Hyponomenta.  No  inulase  was  present  in  the  larvae 
of  these  species  after  they  had  ceased  eating,  nor  in  the  pupae  and 
imagines. 

The  results  of  Kobert  (304)  in  1903,  with  extracts  of  May  beetles, 
cross  spiders,  scorpions,  cockroaches,  ascarides,  pupae  of  pine  spiders, 
and  house  flies,  were  entirely  negative;  so  also  have  been  the  experi- 
ments in  vitro  with  digestive  juices  of  higher  animals,  as  shown  by 
table  on  following  page. 

DIGESTION  AND  UTILIZATION  BY  ANIMALS. 

Inulin  is  hydrolyzed  by  very  dilute  acid  (0.05-0.2  per  cent  at  40° 
C.  according  to  Chittenden),  so  that  its  more  or  less  complete 
inversion  by  the  gastric  juice  is  possible,  and  has  led  many  to  believe 
that  in  spite  of  the  negative  results  obtained  with  amylolytic  enzymes 
shown  above,  it  might  be  converted  into  levulose,  and  as  such  be  read- 
ily utilized  by  the  animal  organism.  It  has  therefore  frequently 
been  recommended  for  the  diet  of  diabetics,  who  show  a  special  tol- 
erance for  levulose;  in  fact,  simply  because  inulin  did  not  reappear  in 
the  urine  as  sugar,  when  fed  to  diabetics,  its  utilization  has  been  as- 
sumed by  many,  no  account  being  taken  of  its  possible  reappearance 
in  the  faeces.  This  reappearance  is  well  demonstrated  in  an  experi- 
ment of  Sandmeyer  (317)  in  which,  after  feeding  80  grams  of  inulin 
to  a  diabetic  dog,  over  46  grams  were  recovered  in  the  faeces. 


298 


Mary  Dames  Swartz, 


AUTHORITY. 

DATE. 

SOURCE  OF  ENZYME. 

KIND  OF 
LEVULAN. 

RESULT. 

Komanos  (303)  

1875 

Saliva 

Inulin 

— 

Pancreatic  juice 

Inulin 

— 

Schmiedeberg  (318; .... 

1879 

Saliva 

Sinistrin 

Chittenden  (292)  

1898 

Saliva 

Inulin 

— 

Pancreatic  juice 

Inulin 

— 

Bierry  and  Portier(288) 

1900 

Macerated  pancreas  and 

intestines  of  dog,  rabbit 

and  seal 

Inulin 

• 

Bierry  andPortier  (288) . 

1  nnn 
1900 

Macerated  pancreas  and  in- 

testines of  dogs,  rabbits; 

fed  three  months  on  arti- 

chokes to  induce  formation 

oi  an  inulase* 

Inulin 

TT„  Jo,,  /9Al\ 

1  nm 
19U1 

Saliva 

Lrraminin 

Bierry  (28bj  

1905 

Pancreatic  juice  of  dog 

Inulin 

Pancreatic  juice  of  dog  + 

macerated    intestines  of 

dogs  and  rabbits 

t  i; 

Inulin 

1910 

Pancreatic  juice  of  dog  from 

pancreatic  fistula  after  in- 

jection of  secretin 

Inulin 

Same  pancreatic  juice  added 

to  macerated  intestines  of 

dog  and  rabbit,  in  slightly 

acid,  slightly  alkaline  and 

neutral  solutions 

Inulin 

Hepato-pancreatic  juice  of 

Helix  pomatia 

Inulin 

Levulose 

Enzyme  prepared  from  he- 

pato-pancreatic  juice  of 

Helix  pomatia 

Inulin 

Levulose 

Weinland  (326)  

1905 

Extract  of  small  intestine  of 

dog 

Inulin 

*Cf.  Richaud,  (326). 


Attempts  to  induce  glycogen  formation  in  rabbits  have  not  justi- 
fied the  hopes  of  the  dieto-therapists  in  regard  to  inulin  as  a  food  for 
diabetics.  The  earlier  experiments  were  either  negative  or  open  to 
criticism  on  account  of  faulty  technique.  The  more  discriminating 
work  of  recent  investigators  (Miura  [313];  and  Mendel  and  Naka- 
seko  [312]),  has  shown  that  little  glycogen  is  formed  from  inulin,  even 
under  the  most  favorable  circumstances.  A  brief  survey  of  the  expe- 
riments in  this  field  is  given  in  the  following  table: 


«_  be 

o  <u 

.2  o 

•°  7 


rd 

2 

bp 

13 

nd 

P 

o 

& 

o 

-*-> 

an 

d 

a 

ure 

less 
ding 

inu 

ho 

<d 

>>  <u 

<u 

<u 

Ui 

enc 

dte 

1 

•   a  ^ 


si    rf  2 


2  d  a 

d  a 

a  d  ri 

os  ;d 
•d  ■*-> 

en 

<L>  en 

a  a  .5 

°  d 

d 


5  g 


I  1 


io  a, 

>  LO 
tn  O 


S  CO 
o  LO 


lo  io  O  rt<  <M  Tt< 

W  N  00  W  O  O  N 

00  ^  IN  MM  CO  H 

o  o  o  o  o  o  o 

H  (N  CO  t)J  lO 


d  .a 

5§ 


1  00C0C001CDNOH 
McONH^tOOOiOH 
<MT*H(MioOOO>OTf( 


<M         lO  1> 
©  ©  ©  ©  O  O 


a  a 


u 

J*3 

u 

u 

V 

'.s 

Js 

LO 

CO 

co 

d 

d 

d 

gms. 

ions 

gms. 

ions 

gms. 

ions 

o 

LO 

o 

to 

o 

LO 

T-H 

22 


a  -5  «> 

cfl  O 

LO  d  'C 

(M   o  <u 


CO  00 
CO 

00 


LO  LO 

00  00 


CO 

o 

CO 

 N 

«  !S 

o  © 
d  3 

a  i2 

o  ^3 


s 

§ 

tg  (324) 

:ericl 

d 

o 
> 

300 


Mary  Dames  Swartz, 


Excluding  the  experiment  of  Luchsinger  (310)  which  was  estimated 
on  a  very  low  specific  rotation  for  glycogen,  only  four  out  of  the  17 
experiments  before  Miura's  (313)  are  positive,  and  in  these  the  gly- 
cogen was  estimated  without  purification,  so  that  the  figures  are  prob- 
ably high.  In  more  reliable  experiments  of  Miura  (313),  and  Mendel 
and  Nakaseko  (312),  the  glycogen  content  of  the  rabbits'  livers  was 
as  low  or  lower  than  the  starvation  maximum  for  the  rabbit,  as  esti- 
mated by  Kulz  (309),  so  that  glycogen  formation  from  inulin  must  be 
regarded  as  doubtful,  or  very  slight. 

When  inulin  is  introduced  parenterally  into  the  organism,  there  is 
no  inversion  or  utilization,  as  shown  by  the  experiments  of  Mendel 
and  Mitchell  (311).  They  injected  warm  solutions  into  the  peritoneal 
cavity,  and  determining  the  output  of  inulin  in  the  urine  (which  was 
sugar-free)  by  calculations  from  the  specific  rotation,  recovered  2.2 
grams  of  2.8  grams  injected.  In  an  experiment  in  which  the  sugar- 
free  urine  was  hydrolyzed,  and  the  output  of  inulin  calculated  from 
the  amount  of  reducing  sugar  obtained,  1.43  grams  were  recovered 
out  of  2.2  grams  injected.  Weinland  (326)  after  subcutaneous  injec- 
tions of  inulin  into  dogs,  continued  for  a  month,  found  no  inulase 
produced  thereby.  On  the  other  hand,  Saiki  (316)  succeeded  in  pro- 
ducing a  definite  anti-inulase  in  rabbit's  serum. 

We  see,  therefore,  that  inulin  is  not  attacked  by  animal  enzymes,  as 
far  as  investigated,  with  the  possible  exception  of  two  species  of  inver- 
tebrates; and  by  a  very  few  vegetable  enzymes.  It  appears  to  a  con- 
siderable extent  in  the  faeces  after  being  fed  per  os  in  spite  of  the  abil- 
ity of  the  gastric  juice  to  hydrolyze  it.  In  spite  of  the  accepted  fact 
that  levulose  is  capable  of  being  directly  utilized  by  the  animal  body 
there  is  no  conclusive  evidence  of  glycogen  formation  from  inulin. 
Whether  other  levulans  resemble  this  hemicellulose  in  these  respects 
has  not  been  investigated. 

Occurrence  and  Nature  of  Dextrans. 

In  the  higher  plants,  starch,  dextrin,  and  cellulose  occur  almost  to 
the  exclusion  of  other  anhydrides  of  dextrose.  A  few  hemicelluloses 
yielding  dextrose  have  been  described,  however,  such  as  "a-amylam" 
(soluble  in  hot  water)  and  "/3-amylam"  (soluble  in  cold  water),  dis- 
covered by  O 'Sullivan  (343)  in  wheat,  rye  and  barley;  those  in  the 
mucilaginous  extracts  of  flax-seed  and  rleabane,  described  by  Bauer 
(329)  and  Rothenfusser  (345);  and  that  in  Colocasia  antiquorum, 
described  by  Yoshimure  (352). 


Nutrition  Investigations. 


301 


Even  in  the  lower  plants,  dextrans  do  not  occur  to  any  great  extent. 
They  have  been  observed  in  bacteria  (338),  yeast  (339),  fungi  (350), 
and  liverworts  (337),  but  occur  most  abundantly  in  lichens  and  algae1 
the  lichens,  as  already  stated,  yielding  dextrans  to  which  the  names 
lichenin,  isolichenin,  usnin,  everniin,  etc.,  have  been  given.  Especial 
interest  is  attached  to  the  dextrans  of  Cetraria  islandica  (lichenin  and 
isolichenin)  which  together  form  80-90  per  cent  of  the  total  carbohy- 
drates of  this  lichen,  because  of  its  abundance  in  northern  lands  and 
its  use  there  as  a  foodstuff;  hence  these  carbohydrates  have  received 
more  attention  from  chemical  investigators  than  any  other  dextrans. 
Ever  since  Berzelius  (333),  in  1808,  studied  the  hot  water  extract  of 
Cetraria  islandica,  and  called  the  carbohydrate  mixture  so  extracted 
"moss-starch,"  on  account  of  its  giving  a  blue  color  with  iodine,  the 
idea  that  it  is,  like  starch,  a  valuable  nutrient,  has  prevailed.  That 
this  hot  water  extract  contained  two  carbohydrates,  one  soluble  in 
cold  water  (isolichenin)  and  the  other  in  hot,  was  demonstrated  by 
Berg  (332)  in  1873,  who  also  showed  that  the  blue  coloration  with 
iodine  was  a  property  of  isolichenin,  but  not  of  lichenin.  Lichenin  was 
first  found  to  yield  dextrose  by  Klason,  in  1886  (337).  The  next  year 
the  two  carbohydrates  were  more  fully  investigated  by  Honig  and  St. 
Schubert  (336),  who  have  carefully  reviewed  the  earlier  literature  on 
this  subject.  That  lichenin  and  isolichenin  yield  dextrose  on  hydroly- 
sis, has  been  verified  by  Karl  Miiller  (341),  Brown  (334),  and  Ulander 
(348),  who  have  also  shown  the  hemicelluloses  of  the  water-insoluble 
part  to  consist  of  dextran,  mannan,  and  galactan,  with  a  small  amount 
of  pentosan.  Escombe's  (335)  observation  that  lichenin  yields  gal- 
actose has  proved  to  be  incorrect. 

DEXTRANASES  IN  THE  VEGETABLE  KINGDOM. 

Honig  and  St.  Schubert  (336)  subjected  isolichenin  to  the  action  of 
malt  diastase,  and  observed  a  rapid  disappearance  of  the  iodine  color 
reaction,  and  the  formation  of  a  dextrin-like  substance  precipitable 
by  alcohol  —  a  result  verified  by  Brown  (334)  in  1898.  Berg  (332) 
treated  lichenin  with  malt  diastase  but  was  unable  to  observe  any 
change  produced  in  it;  his  results  also  have  been  verified  by  Brown 
(334).  The  only  experiments  in  which  sugar  has  been  obtained  from 
lichenin  by  the  action  of  vegetable  enzymes  have  been  carried  out  by 
Saiki  (346)  with  "Taka"  diastase  from  Eurotium  oryzae  and  inulase 
from  Aspergillus  niger. 


*Cf.  p.  255,  also  v.  Lippmann,  Chemie  der  Zuckerarten,  Vol.  I,  pp.  215-220. 


302 


Mary  Davies  Swartz, 


DEXTRANASES  IN  THE  ANIMAL  KINGDOM. 

Attempts  to  hydrolzye  lichenin  by  animal  enzymes  have  been  uni- 
formly unsuccessful.  The  most  exhaustive  researches  were  made  by 
Nilson  (342),  in  1893,  partly  with  pure  lichenin  and  partly  with  the 
powdered  lichen  itself.  Digestions  were  made  with  human  gastric  juice 
for  24  hours,  in  neutral,  acid,  and  alkaline  solutions;  with  pancreatic 
extracts;  with  gastric  juice  followed  by  pancreatic  extract;  and  with 
these  same  extracts,  using  preparations  treated  with  J  per  cent  sodium 
hydroxide  solution  for  24  hours  before  the  digestion.  Nilson  signifi- 
cantly remarks  that  this  resistance  to  sugar-forming  enzymes  is  worthy 
of  note,  inasmuch  as  certain  lichens  have  been  considered  valuable  food 
for  man,  and  that  it  is  hard  to  understand  how  reindeer  utilize  the  car- 
bohydrates of  lichens.  His  negative  results  with  animal  enzymes  have 
been  substantiated  by  Brown  (334) — who  found  digestion  with  0.2  per 
cent  to  0.4  per  cent  hydrochloric  acid  equally  ineffective  —  and  by 
Saiki  (346).  Torup  (347)  reports  that  the  dextran  isolated  from  La- 
minaria  digitata  by  Krefting  is  not  hydrolyzed  by  ptyalin,  amylopsin 
or  diastase. 

DIGESTION  AND  UTILIZATION  IN  ANIMALS  AND  MAN. 

Interest  in  the  digestibility  of  lichenin  arises,  not  only  from  its  use 
in  the  diet  of  normal  individuals,  but  in  the  possibility  of  its  furnish- 
ing a  substitute  for  other  carbohydrates  in  the  diet  of  diabetics. 
After  this  idea  was  set  forth  by  Kiilz  (305),  in  1874,  it  is  not  surprising 
to  find,  in  1879,  the  Italian  physician  Cantani,1  and  the  Norwegian 
physician  Bugge2  reporting  experiments  in  the  use  of  Cetraria  bread 
for  diabetics.  Without  any  further  observations  than  that  the  sugar 
in  the  urine  was  not  increased,  the  idea  prevailed  which  Voit  expres- 
sed in  his  monograph  on  Nutrition  in  1881  (348)  and  Poulsson  repeated 
in  1906  (344),  that  in  some  way  or  other,  the  "moss-starch,"  or 
lichenin,  was  changed  into  sugar  in  the  alimentary  tract,  and  served  as 
a  true  nutrient.  Poulsson  undertook  to  verify  this  by  feeding  experi- 
ments with  two  diabetics,  but  as  Mendel  (340)  has  taken  pains  to 
point  out,  the  results  obtained,  namely  that  45-49  per  cent  of  the  car- 
bohydrates of  the  Cetraria  bread  eaten  were  utilized,  are  unreliable, 
since  the  carbohydrates  of  the  faeces  were  calculated  by  difference, 
instead  of  being  determined  directly  by  analysis. 

iCited  by  Poulsson  (344). 

2Bugge,  Forhandlingar  i  det  medicinske  selskap,  Kristiania,  1879,  p.  179  (cited  by 

Poulsson). 


Nutrition  Investiga lions. 


303 


The  few  feeding  experiments  made  with  animals  do  not  sustain  the 
claims  made  for  the  value  of  Cetraria  as  a  foodstuff.  Brown  (334) 
found  only  1.25-0.7  per  cent  glycogen  in  the  livers  of  rabbits  after 
Cetraria  feeding,  but  these  results  are  not  very  satisfactory,  since  the 
rabbits  would  not  eat  it  very  well.  An  old  experiment  by  von  Mering 
(351),  in  which  16  grams  lichenin  were  fed  to  each  of  two  rabbits, 
shows  0.56-0.63  grams  of  glycogen  in  the  liver,  but  Miura  (313)  has 
pointed  out  that  his  glycogen  estimates  were  probably  too  high.  Saiki 
(346)  fed  Cetraria  extract,  containing  2  per  cent  dry  matter,  in  por- 
tions of  292  cc.  and  300  cc.  on  two  successive  days,  to  a  meat-fed  dog. 
The  faeces  of  the  feeding  period  were  marked  off  at  the  beginning  of 
the  Cetraria  diet  by  fine  quartz,  and  at  the  end  by  cork.  Their  com- 
position is  shown  in  the  following  table: 


DIET 

WEIGHT  AIR 
DRY,  GRAMS. 

CARBOHYDRATE. 

AS  DEXTROSE. 

Meat  

2  days 

10 

5.8 

0.68 

Meat  +  Cetraria  extract .  . 

2  days 

15* 

25.8 

3.90 

Meat  

2  days 

5* 

24.5 

1.20 

Meat  

2  days 

6 

3.2 

0.19 

*  Faeces  of  Cetraria  Period. 


The  Cetraria  extract  contained  6.3  grams  carbohydrate  estimated 
as  dextrose,  the  faeces  5.1  grams. 

Feeding  experiments  on  man,  in  which  the  intake  and  output  of 
carbohydrate  have  been  carefully  determined  by  direct  analysis  of 
the  carbohydrate  as  dextrose,  have  recently  been  conducted  in  Pro- 
fessor Mendel's  laboratory.  The  data  have  not  yet  been  published  in 
detail,  but  from  a  preliminary  description  given  by  Mendel  (340) 
is  taken  the  following  report  of  one  experiment* : 


FAECES. 

Weight  Air  Dry. 

CARBOHYDRATE. 

As  Dectrose. 

CETRARIA  FED. 

Grams. 

Grams. 

Per  cent. 

I.  Fore  period  =  3  days  

35 

2.1 

1 

146 

38.0 

56 

80  g.  =  56g. 

as  dextrose 

II.  Fore  period  =  2  days  

68 

6 

4 

Fore  period  =  daily  

34 

6 

2 

Cetraria  period  =  1  day  

53 

24 

13 

20g.  =  14|g 

as  dextrose 

After  period  =  2  days  

29 

6 

2 

*  From  unpublished  experiments  by  Dr.  V.  C.  Meyers,  Sheffield  Laboratory  of  Physlologi- 
ca  1  Chemistry. 


304 


Mary  Davits  Swartz, 


In  this  experiment,  the  Cetraria  islandica  was  carefully  washed, 
extracted  with  a  dilute  solution  of  potassium  carbonate,  to  remove 
the  bitter  principle;  again  thoroughly  washed,  dried  and  ground  to 
a  powder.  This  preparation  contained  72.5  per  cent  carbohydrate  as 
dextrose.  The  carbohydrates  of  the  diet,  throughout  the  experiment, 
were  limited  to  fine  white  bread  and  zwieback,  forms  in  which  they  are 
utilized  in  man  to  98  per  cent.  The  faeces  were  hydrolized  with  dilute 
acid,  and  the  carbohydrates  determined  as  dextrose  by  Allihn's  gravi- 
metric method.  It  is  evident  that  nearly  all  of  the  Cetraria  carbo- 
hydrate escaped  digestion  and  was  recovered  in  the  faeces. 

Through  the  kindness  of  Professor  Mendel,  the  protocol  of  a  similar 
experiment,  by  Mr.  S.  W.  MacArthur,  is  also  reproduced,  in  which 
the  technique  was  practically  the  same  as  described  for  Dr.  Myers's 
experiment. 


PERIODS. 

DIET. 

COMPOSITION  OF  THE  FAECES. 

Cellulose-Free. 

Weight  Weight 
Moist.       Air  Dried. 

Dextrose. 

Dextrose. 

Fore  =  3  days .... 
Mid  =  3  days .... 
After  =  3  days .... 

Meat,  etc. 

Meat  +  Cetraria* 

Meat,  etc. 

Grams.  Grams. 

281     1  90.0 
542     !  149.0 
284     |  87.5 

Per  cent. 
4.4 
27.6 
4.9 

Grams. 

3.96 
34.5* 
4.2 

*  Amount  Cetraria  eaten  =  47  grams,  which  would  be  equivalent  to  34.1  grams  of  dextrose 

in  faeces. 


It  is  evident  that  the  results  of  this  experiment  simply  confirm  those 
of  Dr.  Myers,  and  demonstrate  that  uncooked  Cetraria,  although 
taken  in  a  form  as  favorable  as  possible  for  its  digestion,  is  scarcely 
affected  by  its  passage  through  the  alimentary  canal,  and  must  be 
classed  among  the  indigestible  carbohydrates.  Very  desirable  expe- 
riments on  the  digestibility  of  the  peculiar  carbohydrate  of  Cetraria  — 
lichenin  —  are  also  being  conducted,  which  may  throw  new  light  on 
the  digestibility  of  the  dextrans,  but  at  present  we  certainly  have  no 
grounds  for  assuming  that  this  group  of  hemicelluloses  deserves  to  be 
classed  with  the  true  nutrients;  all  experiments  show  that  they  are 
not  attacked  by  animal  enzymes,  and  are  recovered  unchanged  in  the 
faeces  after  feeding. 

In  conclusion,  attention  may  be  called  to  certain  data  from  Japan- 
ese dietary  studies,  given  by  Oshima  (15),  as  to  the  digestibility  of 


Nutrition  Investigations. 


305 


some  dried  marine  algae,  which  have  not  been  mentioned  in  connec- 
tion with  the  different  classes  of  hemicelluloses.  The  coefficient  of 
digestibility  for  each  species  studied  is  given  in  the  following  table: 


ALGAE  DRIED. 

OTHER  SUBSTANCES  IN  DIET. 

COEFFICIENT  OF 
DIGESTIBILITY 

(Carbohydrates  includ- 
ing crude  fiber). 

Ecklonia  bicyclis  

Shoyu*  and  sugar 

36.2 

Laminaria  sp  

Shoyu 

75.2 

Laminaria  sp  

Shoyu  and  cleaned  rice 

55.0 

Ulopteryx  pinnatifida  

Cleaned  rice,  shoyu,  sugar 

72.3 

Average   67 . 7 

*  Soy-bean  sauce. 


III.    EXPERIMENTAL  PART. 


Introduction. 

The  foregoing  review  has  emphasized  the  limits  of  our  knowledge, 
both  in  regard  to  the  chemical  composition  of  marine  algae,  and  their 
fate  in  the  alimentary  tract  of  men  and  animals,  as  determined  by 
actual  measurement  of  intake  and  output,  and  as  explained  by  the  ac- 
tion of  bacteria  and  enzymes  in  vitro.  Ten  species  of  marine  algae 
have,  therefore,  been  made  the  basis  of  the  present  investigations. 
Eight  of  them  were  Hawaiian  Limu,  obtained,  as  already  stated, 
through  the  kindness  of  Miss  Minnie  Reed,  Science  teacher  in  the  Ka- 
mehameha  Boys'  School,  Honolulu.  They  were  dried  in  the  sun,  with 
the  salt  water  adhering  to  them,  before  shipping  to  America.  The 
other  two  (dulse  and  Irish  moss)  were  easily  obtained  in  our  Eastern 
markets. 

That  the  carbohydrates  of  algae  are  chiefly  hemicelluloses,  is  indi- 
cated by  the  analyses  which  have  already  been  made;  that  in  many 
species,  these  are  to  a  great  extent  water-soluble,  is  also  well  known. 
In  as  much  as  such  soluble  forms  are  thus  particularly  well  adapted 
for  nutrition  investigation  on  account  of  their  freedom  from  all  in- 
crusting  substances,  which  end  to  interfere  with  digestion,  the  present 
studies  have  been  confined  as  far  as  possible  to  them.  Since  it  was 
desirable  to  study  the  different  groups  of  hemicelluloses,  and  man- 
nans  and  levulans  were  not  found  in  the  seaweeds  in  sufficient  quanti- 
ties for  metabolism  experiments,  these  were  obtained  from  other 
sources;  a  mannan  from  salep,  and  a  levulan  (sinistrin)  from  squills 
(Scilla  maritima). 

Other  investigators  in  this  laboratory  are  working  on  a  dextran 
which  would  naturally  be  included  here,  namely  lichenin  from  Cetra- 
ria  islandica;  consequently  no  experimental  studies  on  this  group  of 
hemicelluloses  have  been  made.  In  considering  any  classifications 
of  these  materials,  it  must  be  borne  in  mind  that  most  of  these  carbo- 
hydrates are  more  or  less  complex  in  nature,  and  can  be  grouped  only 
with  reference  to  what  appears  to  be  the  chief  constituent  in  any 
given  case.  The  following  list  comprises  al  the  species  examined, 
arranged  upon  this  plan: 

306 


Nutrition  Invest igations . 


307 


I.  The  Pentosans: 

Dulse  (Rhydomenia  palmata), 

Limu  Lipoa  (Haliseris  pardalis) , 

Limu  Eleele  (Enteromorpha  intestinalis) , 

Limu  Pahapaha  (Ulva  lactuca  laciniata  and  Ulva  fas data). 

II.  The  Galactans: 

Irish  Moss  (Chondrus  crispus), 

Limu  Manauea  (Gracilaria  coronopifolia), 

Limu  Huna  (Hypnea  nidifica), 

Limu  Akiaki  (Ahnfeldtia  concinna), 

Limu  Uaualoli  (Gymnogongrus  vermicularis  americana  and 

Gymnogongrus  disciplinalis) , 
Limu  Kohu  (Asparagospis  sanfordiana), 
Slippery  Elm  (Ulmus  fulva). 

III.  The  Mannans: 

Salep  (Species  of  Orchis  and  Euiophia). 

IV.  The  Levulans: 

Sinistrin  (Urginea  or  S cilia  maritima). 

The  primary  object  of  these  investigations  has  been  to  determine 
the  fate  of  these  substances  in  the  alimentary  canal  of  man,  since  they 
are  all  used  as  foodstuffs  except  sinistrin,  and  are  all  representative 
of  a  large  class  of  materials  so  employed.  The  experiments  con- 
ducted have  been  Chemical,  Bacteriological  and  Physiological 
in  character,  and  each  of  these  phases  will  be  taken  up  separately  in 
turn  in  the  following  pages. 

Chemical  Investigations. 

The  aim  of  the  experiments  was  to  isolate,  identify,  and  pre- 
pare for  bacteriological  and  physiological  experiments,  any  water- 
soluble  carbohydrates  present  in  sufficient  amount  in  the  materials 
under  consideration;  and  to  determine  such  of  their  properties  as 
would  facilitate  their  detection,  isolation,  and  quantitative  estimation 
in  these  experiments. 

GENERAL  METHODS. 

All  the  seaweeds,  with  the  exception  of  Irish  moss,  were  washed  re- 
peatedly in  cold  tap  water,  to  remove  salt,  sand,  and  other  foreign 
substances,  and  for  convenience,  dried  by  spreading  in  thin  layers 


308 


Mary  Davies  Swartz, 


over  steam  radiators.  The  Irish  moss,  being  comparatively  free  from 
salt,  etc.,  and  largely  soluble  in  pure  water,  was  quickly  washed  once, 
and  extracted  immediately. 

All  hydrolyses  of  carbohydrates  were  made  with  2  per  cent  hydro- 
chloric acid,  by  boiling  with  a  reflux  condenser  over  a  free  flame. 
After  cooling,  the  acid  was  neutralized  with  potassium  hydroxide, 
using  phenolphthalein  as  an  indicator,  when  the  solutions  were  suf- 
ficiently light  in  color;  in  other  cases,  litmus  paper  was  employed. 
When  the  products  of  hydrolysis  served  to  determine  the  nature  of 
the  carbohydrates,  they  were  evaporated  on  a  water  bath  nearly  to 
dryness,  the  residues  extracted  with  hot  95  per  cent  alcohol  the  alcohol 
removed  from  the  filtered  solution  by  evaporation,  the  residues  fre- 
quently taken  up  in  a  little  water  and  decolorized  with  charcoal,  con- 
centrated, and  again  extracted  with  absolute  alcohol. 

All  qualitative  tests  for  reducing  sugar  were  made  with  Fehling's 
solution;  all  quantitative  tests  by  Allihn's  gravimetric  method  for 
dextrose,  the  results  being  calculated  as  dextrose  in  view  of  the  com- 
plex nature  of  most  of  the  products,  and  the  advantage  of  uniformity. 
On  all  preparations  used  for  feeding  experiments,  the  length  of  time 
in  which  the  maximum  yield  of  sugar  could  be  obtained  has  been  de- 
termined, as  a  criterion  in  analyses  of  faeces.  Five  grams  of  dry  air 
material  were  hydrolyzed  in  500  cc.  of  2  per  cent  hydrochloric  acid, 
50  cc.  being  removed  at  intervals  of  one  or  more  hours,  cooled,  neu- 
tralized, made  up  to  100  cc.  and  reducing  power  determined  as  dex- 
trose by  Allihn's  gravimetric  method. 

Tests  for  the  presence  of  fermenting  sugars  have  been  made  in 
fermentation  tubes  with  fresh  compressed  yeast,  using  as  controls 
solutions  of  the  substance  to  be  tested,  without  yeast,  and  dextrose 
solutions  with  yeast. 

All  carbohydrate  solutions  for  polariscopic  examination  have  been 
clarified  by  addition  of  an  equal  volume  of  alumina  cream. 

Qualitative  tests  for  pentosans  have  been  made  by  boiling  the  sub- 
stance to  be  tested  in  a  small  Erlenmyer  flask  with  12  per  cent  hydro- 
chloric acid  and  testing  for  furfurol  with  anilin-acetate  paper. 

Quantitative  tests  for  pentosans  have  been  made  by  the  furfurol- 
phloroglucin  method.1 

Tests  for  galactans  or  galactose  have  been  made  by  oxidation  with 
nitric  acid  to  mucic  acid,  and  the  mucic  acid  identified  by  its  melting 
point  (212°  C.-215°  C). 


lDescribed  in  "Official  and  Provisional  Methods  of  Analysis,"  United  States 
Department  of  Agriculture,  Bureau  of  Chemistry,  Bull.  No.  107,  1907. 


Nutrition  Investigations . 


309 


Qualitative  tests  for  mannose  have  been  made  by  Storer's  (271) 
method.  The  products  of  hydrolysis,  freed  from  the  greater  part  of 
the  salts,  gums,  etc.,  in  the  manner  already  described,  were  taken 
up  in  a  little  water,  and  portions  of  1  cc.  or  2  cc.  placed  in  test 
tubes.  The  reagent  for  testing  was  freshly  prepared  by  shaking 
together  1  cc.  of  phenylhydrazin,  2  cc.  of  glacial  acetic  acid,  and 
10  cc.  of  distilled  water.  3-16  drops  of  this  reagent  were  added  to 
each  of  the  test  tubes,  and  after  standing  several  hours  at  room  tem- 
perature, they  were  examined  for  precipitates  of  mannose-hydrazone. 
These  precipitates  were  examined  under  the  microscope,  because  they 
usually  contained  considerable  amorphous  matter.  The  mannose- 
hydrazone  itself  does  not  come  down  as  colorless  rhombic  plates  at 
first,  but  as  globules  of  greenish-yellow  or  brownish-yellow  color, 
sometimes  smooth  and  resembling  large  yeast  cells  in  the  way  they 
c  uster  together,  and  at  other  times  covered  with  blunt  points  or 
spines.  When  these  globules  were  observed,  the  precipitate  was  care- 
fully washed  with  water,  sometimes  without  removing  from  the  test- 
tube,  the  last  drops  being  taken  up  with  filter  paper,  and  then  dissolved 
in  warm  diluted  alcohol  (3  parts  of  95  per  cent  to  1  part  water),  which 
was  not  filtered,  but  decanted  from  the  amorphous  insoluble  portion, 
and  allowed  to  evaporate  slowly  to  facilitate  the  formation  of  crys- 
tals. Unless  these  crystals  could  be  obtained,  the  tests  were  consid- 
ered negative,  although  Storer  has  pointed  out  that  they  are  sometimes 
difficult  to  obtain,  even  when  true  mannose-hydrazone  balls  are 
present. 

All  quantitative  determinations  have  been  made  in  duplicate  un- 
less otherwise  stated. 

PENTOSAN  PREPARATIONS. 

Dulse. 

A  pure,  water-soluble  pentosan-preparation  has  been  obtained  from 
dulse  (Rhodymenia  palmata).  After  boiling  in  water,  in  an  open 
vessel,  with  occasional  stirring,  for  several  hours,  this  dark,  reddish- 
brown  seaweed  yielded  a  carbohydrate,  non-mucilaginous  in  character, 
which  could  be  precipitated  from  its  solutions  by  alcohol.  About  12 
hours'  boiling  proved  to  be  necessary  for  complete  extractions.  The 
hot,  brown,  watery  extract  was  first  filtered  through  gauze,  and  then 
through  cotton,  as  it  clogged  up  filter  paper  very  quickly.  This 
filtrate,  concentrated  to  a  syrup  on  a  water  bath,  was  poured  while 


310 


Mary  Dames  Swartz, 


still  warm  into  about  three  times  its  volume  of  acetone,  which  expe" 
rience  showed  to  be  a  more  satisfactory  precipitant  than  alcohol* 
Most  of  the  carbohydrate  came  down  very  soon,  in  large,  flocculent, 
yellowish-white  masses,  but  a  portion  remained  in  suspension  as  a 
fine  white  powder,  which  made  filtration  difficult.  The  bulk  of  the 
precipitate  was  therefore  removed  by  filtering  through  three  or  four 
thicknesses  of  fine  gauze,  and  the  rest  obtained  by  distilling  off  the  ace- 
tone, concentrating  the  residue,  and  reprecipitating  the  carbohydrate 
in  solution  with  acetone.  This  precipitate  was  very  hydroscopic,  and 
was  therefore  transferred  immediately  to  95  per  cent  alcohol.  This 
was  replaced  by  fresh  alcohol  after  a  few  hours,  and  the  whole  boiled 
on  a  reflex  condenser  for  half  an  hour.  A  yellowish,  granular  powder 
was  thus  obtained,  which  was  filtered,  washed  with  ether,  and  the  ad- 
herent ether  allowed  to  evaporate.  It  was  then  redissolved  in  a  small 
volume  of  water,  filtered  hot  through  paper,  on  a  jacketed  funnel, 
reprecipitated  with  acetone,  again  put  into  95  per  cent  alcohol,  and 
finally  into  absolute  alcohol,  in  which  it  was  allowed  to  stand  for 
several  weeks.  It  was  then  filtered  off,  washed  with  ether,  and  dried  in 
vacuo  over  sulphuric  acid.  The  product  was  a  cream-white  powder, 
and  apparently  not  at  all  hydroscopic.  From  about  two  kilograms  of 
crude  commercial  dulse,  approximately  75  grams  of  this  material  were 
obtained,  and  used  subsequently  for  feeding  experiments. 

An  attempt  made  to  remove  the  dark  red  coloring  matter  by  extrac- 
tion with  1  per  cent  sodium  carbonate,  led  to  the  discovery  that  this 
carbohydrate  is  readily  extracted  by  dilute  alkaline  solutions.  For 
preparations  on  a  large  scale,  it  was  therefore  found  more  satisfactory 
to  use  the  following  method,  based  on  Salkowski's  method  (139, 140) 
of  obtaining  xylan  and  araban  by  precipitation  with  Fehling's  solu- 
tion. This  method  could  be  applied  exactly  as  described,  but  there 
was  an  evident  tendency  for  the  carbohydrate  to  dissolve  in  the  Feh- 
ling's solution. 

The  dulse  was  accordingly  extracted  with  1  per  cent  potassium 
hydroxide  solution  for  48  hours,  with  occasional  stirring,  the  extract 
removed  by  a  hand  press,  and  the  extraction  with  fresh  alkali  repeated 
for  24  hours.1  These  extracts  were  filtered  through  several  thicknesses 
of  gauze,  and  to  this  filtrate  a  solution  of  copper  sulphate  was  added 
till  the  reaction  was  just  neutral.  A  flocculent,  bluish-green  precipi- 
tate formed.  Into  this  solution  was  stirred  carefully  the  alkaline 
Rochelle  salt-potassium  hydroxide  solution  used  for  Fehling's  solu- 
tion, until  the  precipitate  clumped  together  in  heavy  granular  masses. 


*A  third  extraction  contained  so  little  of  the  material  that  it  was  discarded. 


Nutrition  Investigations. 


311 


This  was  easily  filtered  off  through  gauze,  as  much  liquid  as  possible 
removed  by  pressure,  and  the  precipitate  washed  quickly  with  a 
little  water  to  remove  the  excess  of  alkali.  The  carbohydrate  was 
freed  from  its  copper  compound  just  as  described  by  Salkowski  (140). 
The  precipitate  was  placed  in  a  mortar  and  rubbed  to  a  cream  with 
diluted  hydrochloric  acid  (1  volume  of  water  to  1  volume  of  concen- 
trated acid)  the  acid  being  added  until  all  blue  particles  had  disap- 
peared. It  was  then  poured  into  90  per  cent  alcohol,  the  precipitate 
filtered  off  upon  plaited  paper  and  washed  with  50  per  cent  alcohol, 
replaced  in  90  per  cent  alcohol  acidified  with  hydrochloric  acid,  and 
allowed  to  stand  several  hours  to  dissolve  out  the  copper.  It  was  then 
filtered,  dissolved  in  dilute  potassium  hydroxide,  and  the  dark  brown, 
muddy  solution  filtered  through  paper  on  a  hot  funnel,  the  carbohy- 
drate reprecipitated  with  acid  alcohol,  and  redissolved  and  reprecipi- 
tated  until  free  from  copper.  When  it  no  longer  came  down  readily 
in  alcohol,  acetone  was  substituted,  in  which  it  formed  white  fibrous 
masses  resembling  paper  pulp.  Washed  with  absolute  alcohol  and 
ether,  and  dried  in  vacuo  over  sulphuric  acid,  it  became  a  cream- white 
powder.  Both  of  these  methods  yielded  a  product  readily  soluble  in 
cold  water,  forming  a  clear,  limpid,  amber-colored  solution.  It  gave 
no  color  reaction  with  iodine,  and  contained  no  reducing  substance. 
In  Fehling's  solution  it  formed  a  very  flocculent  white  precipitate,  was 
not  precipitable  by  lead  acetate,  neutral  or  basic,  in  neutral  solution, 
but  formed  a  precipitate  in  alkaline  solutions.  A  test  for  mucic  acid 
gave  negative  results,  but  a  strong  furfurol  reaction  was  obtained  on 
boiling  with  hydrochloric  acid,  indicating  the  presence  of  pentosans. 
A  1-gram  sample  of  material,  prepared  by  the  method  first  described, 
was  tested  quantitatively  for  pentosans.  It  contained  26.8  per  cent 
moisture,  and  2.48  per  cent  ash,  and  yielded  0.076  grams  of  phloro- 
glucid,  from  which  the  yield  of  pentosans,  according  to  Krober's 
tables,1  is  calculated  as  72  per  cent.  The  phoroglucid  precipitates  were 
afterwards  extracted  with  95  per  cent  alcohol,  according  to  Ellett 
and  Tollen's2  method  for  quantitative  determination  of  methyl-fur- 
furol.  The  Gooch  crucibles  containing  the  precipitates  were  warmed 
10  minutes  to  60°  C.  with  15-20  cc.  of  alcohol,  the  extract  filtered  off, 
and  the  extraction  repeated  till  the  alcohol  was  colorless.  The  pre- 
cipitates were  then  dried  at  100°  C.  and  weighed.  The  loss  of  weight 
was  0.0047  grams  or  6  per  cent  of  the  original  precipitate.  The  dulse 
preparation  therefore  contained  a  small  amount  of  methyl-pentosan 


^eitschrift  fur  physiologische  Chemie,  XXXVI,  appendix. 

2Berichte  der  deutschen  chemischen  Gesellschaft,  Vol.  38,  p.  492  (1905). 


312 


Mary  Dames  Swartz, 


The  products  of  hydrolysis  were  tested  for  fermenting  sugar,  with 
negative  results,  but  after  heating  with  phenyl-hydrazin-hydrochloride 
and  sodium  acetate,  an  abundant  yield  of  osazones  was  obtained. 
These  crystallized  out  only  on  cooling,  were  pale  yellow,  soluble  in 
hot  water  only  with  great  difficulty,  but  very  soluble  in  alcohol, 
acetone,  or  pyridin.  After  four  or  five  recrystallizations  from  alcohol, 
they  melted  at  152°  C.  and  this  melting  point  remained  constant  after 
ten  or  twelve  recrystallizations.  However,  there  were  very  minute 
points  at  which  melting  seemed  to  occur  about  140°  C.  Under  the 
microscope,  clusters  of  long  needles  were  seen,  each  with  a  tuft  of 
small  tine  needles  springing  from  its  very  tip.  Dissolved  in  glacial 
acetic  acid,  and  examined  in  a  100  mm.  tube,  these  osazones  showed 
no  rotation  of  polarized  light. 

A  very  white  sample  of  the  dulse  carbohydrate  was  used  to  deter- 
mine its  specific  rotation.  It  contained  7.1  per  cent  moisture  and 
1.68  per  cent  ash.  Two  determinations  were  made,  one  on  a  0.6  per 
cent  solution  and  the  other  on  a  1.0  per  cent  solution  for  which  the 
polariscope  readings  in  a  200  mm.  tube  were  respectively  —0.90° 
and  —1.52°.  The  specific  rotation,  calculated  from  these  readings 
was  therefore  [a]D  =  —75.2°  and  —76.2°,  or  corrected  for  moisture 
and  ash,  [o]D  =  -82.4°  and  -83.6°,  average,  -83°. 

The  rate  of  hydrolysis  and  maximum  reducing  power  were  deter- 
mined as  follows:  5  grams  of  the  material  dissolved  in  500  cc.  of  2 
per  cent  hydrochloric  acid  were  boiled  in  the  usual  way.  At  the  end 
of  two  hours,  and  at  intervals  of  one  hour  thereafter,  50  cc.  portions 
were  removed,  neutralized  and  made  up  to  100  cc,  and  the  amount 
of  reducing  sugar  present  determined  as  dextrose.  The  following 
results  were  obtained: 


That  the  results  vary  greatly  with  the  concentration,  is  shown  by 
the  fact  that  a  0.3  per  cent  solution  boiled  5  hours  yielded  67.1  per 
cent  of  sugar  as  dextrose. 

Having  established  the  fact  that  this  dulse  preparation  consists  of 
pentosans,  with  the  properties  described,  further  investigations  into 
the  exact  chemical  nature  of  the  carbohydrates  composing  it  were 
not  considered  within  the  province  of  this  work. 


TIME  OF  BOILING. 


SUGAR  AS  DEXTROSE. 


Hours. 


2 
3 
4 
5 


Per  cent. 

87.2 
87.2 
89.4 
89.5 


Nutrition  Investigations. 


313 


Hawaiian  Seaweeds. 


Beside  the  dulse  preparation,  three  seaweeds  have  been  included  in 
this  group  which  yielded  little  or  no  soluble  carbohydrates,  namely, 
Limu  Lipoa  (Haliseris  pardalis),  Limu  Eleele  (Enter vmorpha  intesti- 
nalis)  and  Limu  Pahapaha  (Ulva  lactuca,  etc.). 

Limu  Lipoa.  Limu  Lipoa  contained  a  small  amount  of  non-muci- 
laginous carbohydrate,  soluble  in  cold  water  as  well  as  hot.  It  was 
precipitated  by  alcohol,  in  which  it  came  down  as  a  white  fibrous 
mass.  On  hydrolysis,  it  yielded  a  dextro-rotatory  fermenting  sugar; 
a  test  with  phenylhydrazin  acetate  for  mannose  was  negative,  as  were 
tests  for  pentosans.  The  total  amount  of  this  carbohydrate  was  so 
small  as  to  be  almost  negligible,  as  far  as  feeding  experiments  were 
concerned,  hence  the  original  washed  material  was  used,  after  grinding 
to  a  powder  in  a  coffee  mill.  It  contained  a  very  high  percentage  of 
inorganic  matter  because  the  thalli  were  so  encrusted  with  calcareous 
substances,  that  it  was  impossible  to  remove  them  entirely  by  washing. 
This  preparation  gave  a  strong  f urf urol  test,  and  a  single  quantitative 
test  for  pentosans  gave  the  following  results: 

The  sample,  weighing  1  gram,  contained  10.5  per  cent  moisture  and 
18.5  per  cent  ash.  It  yielded  0.161  grams  of  phloroglucid,  which 
according  to  Krober's  tables  1  is  equivalent  to  0.147  grams  pentosans, 
or  14.7  per  cent  of  the  crude  substance. 

Tests  for  starch  and  reducing  sugar  were  negative.  Only  a  minute 
quantity  of  mucic  acid  was  obtained;  a  quantity  too  small  to  purify 
and  determine  the  melting  point.  The  products  of  hydrolysis  showed 
slight  fermentation,  which  was  doubtless  due  to  the  mannan  of  the 
water-extract. 

A  determination  of  the  reducing  power  made  in  the  same  manner 
as  already  described,  gave  the  results: 


Limu  Eleele.  Limu  Eleele  yielded  no  appreciable  amount  of  water- 
soluble  carbohydrate,  even  after  boiling  3  or  4  hours.    The  dried 


TIME  OF  BOILING. 


SUGAR  AS  DEXTROSE. 


Hours. 


Per  cent. 
Very  little 


H 
3 
4 
6 

S 


14.3 
14.7 
12.9 
12.8 


1  Zeitschrift  fiir  physiologische  Chemie,  XXXVI,  appendix. 


314 


Mary  Davies  Swartz, 


seaweed  was  therefore  simply  finely  ground  for  use  in  feeding  experi- 
ments. 

It  gave  a  strong  furfurol  test,  but  yielded  a  mere  trace  of  mucic 
acid.  Tests  for  starch  and  reducing  sugar  were  negative.  The 
products  of  hydrolysis  contained  no  fermenting  sugar.  From  this  it 
was  evident  that  the  hemicelluloses  were  chiefly  pentosans. 

Determination  of  the  reducing  power  gave  the  following  results: 

TIME  OF  BOILING.  SUGAR   AS  DEXTROSE. 

Hours.  Per  cent. 

2  16.8 

3  16.9 

4  18.1 

5  16.8 

Limu  Pahapaha.  Ulva  lactuca  is  said  by  Rohmann  (134)  to  con- 
tain a  water-soluble  methyl -pentosan,  rhamnosan;  but  if  this  occurs 
in  Limu  Pahapaha,  it  must  be  in  very  small  amount,  as  an  extract 
of  50  grams  of  the  dried  seaweed,  made  by  boiling  3  or  4  hours,  gave 
very  little  residue  on  evaporation  to  dryness.  For  feeding  experi- 
ments, the  dry  crude  substance  was  simply  ground  to  a  powder. 
Like  Limu  Eleele,  it  gave  a  strong  furfurol  test,  but  yielded  no  mucic 
acid.  Starch  was  present,  but  no  reducing  sugar.  Fermentation 
with  yeast  was  marked  in  12  hours,  probably  due  chiefly  to  the  hy- 
drolysis of  the  starch. 

Determination  of  reducing  power  gave  the  following  results: 

TIME  OF  BOILING.  SUGAR  AS  DEXTROSE. 

Hours.  Per  cent. 

2  28.8 
4  31.8 


GALACTAN  PREPARATIONS. 


Irish  Moss. 


The  carbohydrates  of  Irish  moss  are,  as  already  noted,  readily 
soluble  in  cold  water,  after  the  salt  has  been  removed  from  the  sea- 
weed. By  allowing  the  moss  to  stand  for  24  hours  in  cold  water  (about 
10  liters  to  250  grams  of  dry  substance),  an  almost  colorless,  semi- 
transparent,  mucilaginous  extract  was  obtained.  By  straining  this 
off  through  gauze,  and  allowing  it  to  stand  over  night,  for  minute 
particles  of  cellulose  held  in  suspension  to  settle,  a  solution  almost 
entirely  free  from  insoluble  material  was  obtained  by  decantation. 


N  utrition  Investigations. 


315 


This  was  considered  sufficiently  pure  for  feeding  experiments,  and  was 
quickly  dried  by  pouring  into  broad  shallow  dishes  and  placing  over  a 
steam  radiator.  It  formed  yellowish,  translucent  scales,  which  were 
easily  removed,  and  finely  ground. 

Subsequent  extractions  were  made  in  a  steam  sterilizer,  heating 
several  hours  at  a  time.  Tests  showed  that  the  carbohydrate  was 
not  hydrolyzed  by  this  repeated  subjection  to  high  temperature. 
The  several  extracts  were  first  strained  off  through  gauze  and 
then  filtered  hot  through  cotton,  to  remove  the  cellulose 
particles.  As  these  clogged  even  cotton  filters  very  rapidly,  it 
was  found  most  satisfactory  to  let  the  extracts  stand  over  night, 
decant  off  the  supernatant  fluid  as  far  as  possible,  and  filter 
in  a  water-jacketed  funnel.  Solutions  containing  over  1  per  cent 
dry  substance  could  not  be  filtered  through  paper.  For  experi- 
ments where  a  perfectly  clear  fluid  was  desired,  a  \  per  cent 
solution  was  filtered  hot  through  plaited  paper,  and  then  concentrated 
on  a  water  bath  to  the  desired  strength.  One  per  cent  solutions 
formed  a  soft  jelly  on  cooling;  2  per  cent  solutions,  a  firm  jelly. 

Even  when  evaporated  to  a  thick  syrup,  the  carbohydrates  of  the 
Irish  moss  extract  are  not  readily  precipitated  by  comparatively  large 
volumes  of  95  per  cent  alcohol,  but  form  a  voluminous,  transparent, 
gelatinous  mass.  This  was  found  to  be  more  or  less  characteristic  of 
all  the  galactans  examined.  They  could  be  brought  down  most 
satisfactorily  by  addition  of  sodium  chloride  to  the  extract  before 
pouring  it  into  the  alcohol.  In  this  way  a  white  precipitate  of  fine 
fibers  was  obtained  from  the  moss.  The  carbohydrate  could  also 
be  precipitated  by  saturation  with  potassium  acetate,  and  freed  from 
inorganic  salts  by  dialysis,  according  to  the  method  described  by 
Pohl  (263).  It  could  not  be  precipitated  by  Fehling's  solution,  nor 
by  lead  acetate  in  neutral  solution. 

Owing  to  the  opacity  of  its  solutions,  and  to  the  fact  that  its  gelat- 
inizing property  made  the  use  of  very  dilute  solutions  necessary,  no 
satisfactory  determination  of  its  specific  rotation  could  be  obtained. 
A  0.5  per  cent  solution,  clarified  with  alumina  cream,  and  examined  in 
a  200  mm.  tube,  showed  a  rotation  of  +0.34°,  and  other  trials  gave 
positive  evidence  that  it  was  dextro-rotatory.  The  products  ot  hydro- 
lysis were  also  dextro-rotatory,  and  yielded  osazones,  which  after  one 
recrystallization  from  alcohol,  had  a  melting  point  of  184°-185°  C. 

The  carbohydrate  gave  a  red- violet  color  with  iodine,  and  con- 
tained no  reducing  sugar.  A  faint  furfurol  test  was  obtained.  Oxi- 
dation with  nitric  acid  gave  a  rich  yield  of  mucic  acid.    Since  Hadike, 


316 


Mary  Dames  Swartz, 


Bauer  and  Tollens  (185),  and  Miither  (200)  have  already  shown  that 
Irish  moss  contains  galactan,  levulan,  dextran  and  pentosan  groups, 
these  tests  were  simply  verifications  of  some  of  their  observations. 
Determination  of  the  reducing  power  gave  the  following  results: 

TIME   OF  BOILING.  SUGAR  AS  DEXTROSE. 

Hours.  Per  cent. 

2  45.6 

3  48.6 

4  45.8 

I 

Hawaiian  Seaweeds. 


Limu  Manauea  (Gracilaria  cor  onopif alia), 
LimuHuna  (Hypnea  nidifica), 
Limu  Akiaki  (Ahnfeldtia  concinna), 
Limu  Kohu  (Asparagopsis  sanfordiana), 
Limu  Uaualoli  (Gymnogongrus) . 

k  These  five  seaweeds  all  contained  soluble  carbohydrates,  which  were 
extracted  by  boiling  in  water  in  an  open  vessel  over  a  free  flame  for 
two  hours  or  longer.  Limu  Manauea,  Limu  Huna,  and  Limu  Akiaki, 
which  consist  largely  of  soluble  gelatinizing  hemicelluloses,  yielded 
most  of  these  on  boiling  two  or  three  hours.  The  extracts  were  strained 
off  through  gauze,  filtered  hot  through  cotton,  and  dried  in  thin 
sheets  as  described  for  Irish  moss.  While  the  preparations  were  dark 
colored,  and  had  a  decided  "sea"  flavor,  they  were  not  unpleasant, 
and  were  used  in  feeding  experiments  without  further  purification. 
As  already  stated,  the  carbohydrates  were  not  easily  precipitated  with 
alcohol  unless  a  neutral  salt  (as  sodium  chloride)  was  present. 

Limu  Kohu  and  Limu  Uaualoli  contained  only  a  small  proportion 
of  soluble  hemicelluloses,  and  this  was  obtained  only  after  boiling 
8  to  24  hours.  The  extracts  were  also  much  less  gelatinous  in  charac- 
ter. The  thalli  of  Limu  Kohu  are  almost  like  wire  when  dry,  and 
remain  tough  and  hard  even  after  many  hours'  boiling.  The  extracts 
of  these  two  species  were  more  readily  precipitated  by  alcohol  than 
the  others,  but  the  precipitation  was  greatly  facilitated  by  adding 
sodium  chloride.  The  carbohydrate  of  Limu  Kohu  was  precipitated 
as  a  white  cheese-like  cake,  floating  on  the  surface,  while  that  of  Uaua- 
loli came  down  as  a  mass  of  coarse  white  fibers.  These  precipitates 
were  transferred  to  absolute  alcohol,  and  after  standing  several  days, 
were  filtered  off,  washed  with  ether  and  dried  at  40°-50°  C.  The 


Nutrition  Investigations.  317 

Kohu  preparation  should  have  been  dried  in  vacuo,  for  it  proved  to 
be  slightly  hydroscopic,  and  instead  of  remaining  a  fine  white  powder, 
became  somewhat  brownish.  The  Uaualoli  preparation  dried  easily 
to  a  grayish  white,  light,  fibrous  mass. 

Tests  for  starch  and  reducing  sugar  were  negative  on  all  these 
substances.  Tests  for  galactans  and  pentosans  were  positive  in  every 
case.  Three-gram  samples  of  the  air-dry  preparations  of  Limu 
Akiaki,  Limu  Uaualoli  and  Limu  Kohu  respectively  yielded  0.53 
grams,  0.92  grams  and  0.64  grams  of  mucic  acid,  recrystallized  once 
from  ammonium  carbonate.1  The  products  of  hydrolysis  in  no  case 
contained  fermenting  sugars.  It  is  evident  therefore,  that  these  five 
preparations  from  the  foregoing  Hawaiian  seaweeds  consisted  chiefly 
of  galactans^  accompanied  by  some  pentosan-groups.  From  the 
frequency  with  which  methyl-pentosans  have  been  shown  to  occur 
in  all  seaweeds  previously  investigated,  it  is  very  likely  that  they 
occur  in  all  these  varieties  and  it  would  be  desirable  to  make  tests  for 
methyl-pentosans. 

Determinations  of  the  reducing  power  were  made,  as  shown  in  the 
following  table: 


SPECIES  OE  SEAWEED. 

SUGAR  AS  DEXTROSE. 

1  Hour. 

2  Hours. 

3  Hours. 

4  Hours. 

Per  cent. 

Per  cent. 

Per  cent. 

Per  cent. 

Limu  Manauea  

41.9 

44.6 

39.8 

Limu  Huna  

43.6 

58.4 

55.6 

30.8 

Limu  Akiaki  

36.0 

36.0 

34.0 

Slippery  Elm.  For  the  preparation  of  the  carbohydrate  which 
forms  the  mucilaginous  extract  of  slippery  elm  bark,  pieces  of  the 
latter  were  torn  into  narrow  strips  and  allowed  to  stand  over  night 
in  cold  water,2  and  then  the  mucilage  expressed  by  squeezing  through 
gauze.  This  process  was  repeated  until  the  bark  became  a  mass  of 
separate  fibers.  The  mucilaginous  principle  swells  in  cold  water  to 
a  transparent  jelly,  but  is  soluble  only  to  a  very  limited  extent.  It 
was  found  impossible  to  filter  it,  even  through  gauze,  and  therefore, 
although  it  contained  small  particles  from  the  disintegrated  bark 


*For  method  cf.  Bull.  No.  107,  p.  55,  Bureau  of  Chemistry,  United  States 
Dept.  of  Agriculture. 

2  It  was  found  impossible  to  extract  the  mucilaginous  principle  in  hot  water. 


318 


Mary  Dames  Swartz, 


fibers,  the  carbohydrate  was  precipitated  by  pouring  the  thick  slimy 
mass  into  about  six  times  its  volume  of  95  per  cent  alcohol.  After 
standing  some  hours,  a  transparent,  gelatinous  precipitate  settled  to 
the  bottom,  and  was  filtered  off  through  several  thicknesses  of  gauze. 
Dehydrated  by  means  of  absolute  alcohol  and  ether,  it  formed  a  gray- 
ish-brown powder.  This  was  found  to  be  soluble  in  dilute  alkali,  and 
was  subsequently  purified  by  dissolving  in  1  per  cent  potassium  hy- 
droxide, filtering  through  cotton  and  reprecipitating  with  95  per  cent 
alcohol.  The  product  was  somewhat  lighter  in  color  than  at  first, 
but  still  far  from  white.  It  was  soluble  in  hot  Fehling's  solution,  but 
precipitable  with  lead  acetate.  It  gave  no  color  with  iodine,  although 
a  small  amount  of  starch  was  present  in  the  original  bark. 

Furfurol  tests  were  faint  showing  only  traces  of  pentosans,  but  the 
yield  of  mucic  acid  was  large,  0.15  grams  of  mucic  acid  being  obtained 
from  ]  gram  of  the  air  dry  powder. 

The  products  of  hydrolysis  were  dextro-rotatory  and  contained 
no  fermenting  sugars.  Hence  this  preparation  consisted  chiefly  of 
galactan. 

A   MANNAN  PREPARATION. 

Since  none  of  the  algae  which  form  the  basis  of  these  studies  yielded 
mannan,  save  Limu  Lipoa,  and  that  in  amounts  inadequate  for  the 
experiments  proposed,  this  hemicellulose  was  obtained  in  soluble 
form  from  salep.  Both  the  small,  horny  dried  tubers  and  the  grayish- 
white  powder  made  from  them,  werj  purchased  from  Schieffelein&  Co., 
New  York. 

A  preparation  of  pure  mannan  was  made  in  the  following  way: 
The  tubers  were  soaked  in  cold  water  24  hours,  washed  thoroughly 
and  ground  in  a  meat  chopper.  To  this  mass,  cold  water  was  added 
in  large  volume,  and  the  whole  allowed  to  stand  over  night,  then  the 
dissolved  mannan  filtered  off  through  gauze.  According  to  Hilger 
(254),  the  extract  made  in  this  way  should  contain  no  starch.  But 
when  the  tubers  are  heated  before  drying,  the  starch  is  made  soluble, 
and  in  this  instance  the  cold  water  extract  gave  a  blue  color  with 
iodine.1  Hence  subsequent  extractions  were  made  with  hot  water  on 
a  water  bath,  for  several  hours.  The  salep  swells  very  much  in  water 
so  that  a  very  large  portion  was  required  to  get  the  mannan  all  into 


1  Salep  tubers  purchased  since  this  work  was  done  yielded  only  a  trace  of  starch 
;n  the  cold  water  extract. 


Nutrition  I nvestigations. 


319 


solution.1  The  extracts,  strained  through  cheese  cloth,  were  digested 
24  hours  with  malt  diastase  to  free  from  starch,  then  concentrated  to 
a  thick  syrup  on  a  water  bath,  and  poured  into  three  times  their 
volume  of  95  per  cent  alcohol.  A  voluminous,  flocculent,  and  some- 
what fibrous,  snow-white  precipitate  formed,  which  was  filtered  off, 
pressed  free  from  alcohol,  redissolved  in  hot  water,  and  reprecipitated. 
(This  was  done  largely  to  free  it  from  sugar  produced  by  the  diges- 
tion of  the  starch.)  It  was  then  transferred  to  absolute  alcohol  and 
allowed  to  stand  three  or  four, days,  after  which  it  was  washed  with 
ether,  and  dried  in  a  vacuum  desiccator.  A  somewhat  coarse  white 
powder  resulted,  containing  6.94  per  cent  moisture  and  0.74  per  cent 
ash.2  It  swelled  up  very  readily  in  water,  but  dissolved  exceedingly 
slowly  to  a  colorless,  semi-transparent  mucilaginous  solution,  which 
did  not  reduce  Fehling's  solution,  and  examined  in  the  polariscope, 
after  clarification  with  alumina  cream,  appeared  optically  inactive. 
However,  on  reprecipitating  the  carbohydrate  with  alcohol,  and 
examining  the  alcoholic  filtrate,  sugar  was  found  to  be  present  in 
small  amount.  A  solution  absolutely  sugar-free  became  optically 
active.  A  sample  in  which  the  sugar  had  been  removed  by  fermen- 
tation with  yeast,  was  used  to  determine  the  specific  rotation.  The 
following  results  were  obtained:  (1)  A  2  per  cent  solution  in  a  200 
mm.  tube  read  —1.59°;  applying  corrections  for  moisture  and  ash, 
[a]D  =  —43.1°.  (2)  A  sample  containing  in  100  cc.  0.5868  grams 
mannan  dried  to  constant  weight  at  105°  C.  read  —0.48°;  corrected 
for  0.4  per  cent  ash,  [a]D  =  —43.8°.  According  to  Thamm  (276), 
salep  extract  is  inactive.  In  the  above  experiments,  the  levo-rotatory 
nature  of  the  mannan  was  at  first  obscured  by  the  presence  of  traces 
of  reducing  sugar  formed  by  the  hydrolysis  of  the  starch,  which  could 
not  be  detected  by  testing  directly  by  Fehling's  solution.  Thamm, 
however,  in  several  ways  carefully  tested  salep  hydrolysis  products 
for  dextrose  with  negative  results,  so  that  the  only  way  to  account  for 
these  conflicting  results  seems  to  be  to  attribute  it  to  difference  in  the 
specimens  of  Orchis  which  furnished  the  mannan. 

Salep-extract  is  readily  precipitated  by  Fehling's  solution  in  floccu- 
lent white  masses.  It  is  not  precipitated  by  lead  acetate  in  neutral 
solution  (nor,  according  to  Thamm  [276],  in  solutions  of  other  neutral 
salts),  but  is  precipitated  by  basic  lead  acetate. 

A  furfurol  test  was  faintly  positive,  verifying  the  report  of  traces  of 
pentosans  by  Tollens  and  Widtsoe  (163),  and  also  by  Thamm  (276). 


*15  liters  of  water  to  100  grams  salep  powder,  according  to  Thamm  (276). 
2Thamm  found  0.483  per  cent. 


320 


Mary  Dames  Swartz, 


The  products  of  hydrolysis  were  dextro-rotatory  and  contained 
sugar  fermentable  with  yeast.  A  rich  yield  of  mannose-hydrazone 
was  obtained  with  phenyl-hydrazine  acetate,  melting  on  recrystalliza- 
tion  at  188°  C.  According  to  Thamm  (276),  salep  extract  yields  ex- 
clusively mannose  on  complete  hydrolysis. 

Hydrolyzed  for  three  hours,  the  reducing  power  of  this  mannan  was 
91.6  per  cent. 

Determinations  of  ash,  moisture,  starch,  and  mannan  were  made  on 
the  salep  obtained  in  the  form  of  a  powder.  Starch  and  mannan  were 
determined  as  follows:  1  gram  of  air  dry  powder  was  boiled  in  250  cc. 
water,  and  after  cooling  to  37.5°  C,  the  starch  hydrolyzed  with  malt 
diastase,  dialyzed  sugar-free.  The  solution  was  then  filtered,  con- 
centrated to  small  volume,  and  the  mannan  precipitated  with  absolute 
alcohol.  The  precipitate  was  filtered  off,  dissolved  in  a  little  water 
and  reprecipitated,  to  obtain  any  sugar  retained  in  the  first  precipi- 
tation. The  mannan  was  then  dried  at  100°  C.  and  weighed.  The 
filtrates  were  combined,  freed  from  alcohol,  hydrolyzed  with  2  per 
cent  hydrochloric  acid  45  minutes  to  convert  all  the  maltose  to  dextrose, 
and  sugar  determined  by  Allihn's  method.  The  results  of  these  analy- 
ses are  shown  in  the  following  table: 

Per  cent  Per  cent 

Moisture  0.77       Starch  26.4 

Ash  8.9        Mannan  19.5 

According  to  Dragendorf1  the  composition  of  Orchis  tubers  is  as 
follows : 

Per  cent  Per  cent 

Starch   27.3       Protein   4.9 

Mucilage   48.1       Cellulose   2.4 

Sugar   1.2 

Thamm  also  reports  a  yield  of  40-45  per  cent  mucilage  from  the 
salep  powder  used  in  his  investigations.  Hence  the  powder  used  in 
this  the  present  experiment  was  for  some  reason  very  deficient  in 
mannan. 

Its  reducing  power  was  as  follows: 

TIME  OF  BOILING.  SUGAR   AS  DEXTROSE. 

Hours.  Per  cent 

2  74.2 

3  75.8 
5  75.8 


iCited  in  the  National  Dispensatory  (1884),  also  by  Thamm  (276). 


NiUr  it  ion  InVi  'Stig  a  I  ions. 


321 


A  LEVULAN  PREPARATION. 


Commercial  Squills,  consisting  of  the  dried  and  broken  leaves  of 
the  bulbs  of  Scilla  maritima  (or  Urginea  Scilla  Stenh.)  yield,  as  dis- 
covered by  Schmiedeberg  (318),  the  levulan  sinistrin.  They  were 
finely  ground  in  a  coffee  mill,  and  the  sinistrin  prepared  according  to 
Schmiedeberg's  directions.  To  the  dry  powder  sufficient  water  was 
added  to  make  a  thin  cream,  and  then  a  saturated  lead  acetate  solu- 
tion until  further  addition  produced  no  precipitate.  To  the  clear, 
straw-colored  filtrate,  freed  from  lead  with  hydrogen  sulphide,  was 
added  freshly  prepared  milk  of  lime,  with  constant  stirring,  until  a 
somewhat  creamy  consistency  was  produced.  To  facilitate  the  for- 
mation of  sinistrin-calcium  carbonate,  this  mixture  was  concentrated 
on  the  water  bath  for  some  time  (as  suggested  by  Reidemeister)  [314]. 
The  precipitate  was  then  sucked  dry  on  a  Biichner  funnel,  washed 
thoroughly  with  cold  water  (being  rubbed  up  in  a  mortar  for  the  pur- 
pose), again  sucked  dry,  rubbed  to  a  cream  with  water,  and  treated 
with  carbon  dioxide  until  the  fluid  was  no  longer  alkaline  to  litmus. 
After  heating  to  facilitate  the  complete  separation  of  the  calcium 
carbonate,  the  sinistrin  in  solution  was  filtered  off,  a  little  oxalic  acid 
carefully  added  to  remove  the  last  traces  of  lime,  and  the  solution  then 
decolorized  with  charcoal,  and  evaporated  to  a  syrup  at  a  temperature 
of  about  40°  C.  From  this  solution  the  sinistrin  was  precipitated  with 
95  per  cent  alcohol,  as  a  white  gummy  mass.  Transferred  to  abso- 
lute alcohol,  and  allowed  to  stand  24-36  hours  it  became  very  tenacious, 
but  on  longer  standing,  with  occasional  stirring,  it  grew  brittle,  and 
finally  crumbled  to  a  coarse  white  powder,  which  was  dried  in  a 
vacuum  desiccator.  This  material  was  readily  soluble  in  cold  water. 
(According,  to  Schmiedeberg  [318],  even  solutions  of  20-30  per  cent 
are  not  syrup-like.)  It  gave  no  color  with  iodine,  did  not  reduce 
Fehling's  solution,  and  was  not  precipitated  by  it.  This  preparation, 
at  first,  contained  13  per  cent  moisture  and  0.76  per  cent  ash.  De- 
termination of  the  specific  rotation  then  gave  the  following  results: 
A  2  per  cent  solution  in  a  200  mm.  tube,  read  —1.32°;  corrected  for 
moisture  and  ash,  [a]D  =  —38.2°.  After  longer  standing  (three 
months)  over  sulphuric  acid,  the  moisture  content  was  4.8  per  cent, 
and  determination  of  specific  rotation  gave  the  following  results: 
A  1  per  cent  solution  in  a  200  mm.  tube,  read  —0.55°;  corrected  for 
moisture  and  ash,  [a]D  =  —29.1°.  Schmiedeberg  (318)  found  the 
average  for  [a]D  =  —41.4°,  and  Reidemeister  (314),  [a]D  =  —34.6°. 
It  is  impossible  to  account  for  these  differences.   Reidemeister  claims 


322 


Mary  Dames  Swartz, 


that  the  rotation  increases  on  standing,  but  in  these  solutions  there 
was  no  change  in  48  hours,  at  room  temperature. 

On  hydrolysis,  sinistrin  yields  a  levo-rotatory,  reducing  sugar,  fer- 
menting with  yeast,  Schmiedeberg  (318)  reports  this  as  a  mixture  of 
levulose  and  an  inactive  sugar,  but  Reidemeister  (314)  declares  that 
it  is  neither  a  mixture  of  levulose  and  an  inactive  sugar,  nor  of  levu- 
lose and  dextrose,  in  spite  of  the  fact  that  he  found  for  it  |a]D=  —88°, 
while  for  levulose,  [a]D=  —106°,  a  difference  for  which  he  is  unable 
to  account. 

SUMMARY. 

The  composition  of  the  preparations  which  have  been  described  is 
best  shown  in  the  following  table: 


NATURE  OF  CARBOHYDRATES  PRESENT. 


SOURCE  OF  MATERIAL. 

Pentosans. 

Galactan. 

Mannan. 

Levulan. 

Dextran. 

Dulse  (Rhodymenia  Palmata) 

+ 

Limu  Lipoa    {Haliseris  Par- 

+ 

Limu    Eleele    {Enter  omorpha 

intestinalis)  

+ 

Limu   Pahapaha    {Ulva  lac- 

tuca,  etc.)  

+ 

(Starch) 

Irish  Moss  {Chondrus  crispus) 

Trace 

+ 

+ 

+ 

Limu  Manauea  {Gracilaria 

+ 

+ 

Limu  Huna  (Hypnea  nidifica) 

+ 

+ 

Limu  Akiaki  {Ahnjeldtia  con- 

cinna)  

+ 

+ 

Limu    Uaualoli  {Gymnogon- 

grus)  

+ 

+ 

Limu  Kohu  {Asparagopsis 

+ 

+ 

Slippery  Elm  (Ulmus)  

+ 

Trace 

+ 

Squills  (JJrginea  s cilia)  [Sinis- 

trin]   

+ 

The  foregoing  observations  correspond  with  those  of  Konig  and 
Bettels  (8),  in  that  the  marine  algae  all  yield  pentosans,  and  fre- 
quently galactans.  The  gelatinizing  principle  in  every  case  appears 
to  be  due  to  the  galactan  groups.    No  specific  tests  have  been  applied 


Nutrition  Investigations. 


323 


for  fructose,  the  polysaccharide  of  which  also  appears  to  be  common 
in  algae,  but  the  absence  of  fermenting  sugar  in  all  the  algae  except 
Limu  Lipoa,  indicates  that  if  present,  it  is  in  too  small  amount  to  be 
detected  in  the  hydrolysis  products  of  5-10  grams  of  crude  material. 

The  reducing  power  has  been  determined  on  each  substance  used  in 
feeding  experiments;  the  results  of  all  determinations  are  summarized 
in  the  following  table: 


SUBSTANCE. 


SUGAR  AS  DEXTROSE  AFTER  BOILING. 


1  Hour 


2  Hours. 


3  Hours 


4  Hours.  5  Hours 


6  Hours, 


8  Hours. 


Per  cent.  Per  cent. 


Percent. 


Dulse  

Limu  Lipoa  

Limu  Eleele  

Limu  Pahapaha  

Irish  Moss  

Limu  Manauea  

Limu  Huna  

Limu  Akiaki  

Salep  (Powder)  

Salep  (Pure  mannan) 


87 

2 

87 

3 

14 

3 

16 

8 

16 

9 

28 

8 

45 

6 

48 

G 

41 

9 

44 

6 

58 

4 

55 

6 

36 

0 

34 

0 

74.2 


75.8 
91.6 


Per  cent, 
89.4 
14.7 
18.1 
31.8 

45.8 
39.8 
30.6 


Per  cent. 

89.5. 
16.8 


75.8 


Per  cent. 

12.9 


Per  cent. 
12.8 


Bacteriological  Investigations, 
introduction. 

It  is  an  accepted  fact  that  even  cellulose,  with  its  high  powers  of 
resistance,  is  to  some  extent  decomposed  in  the  alimentary  tract  by 
bacteria.  It  is  therefore  reasonable  to  expect  that  the  less  resistant 
hemicelluloses  will  also  be  attacked  and  decomposed  by  bacteria. 
The  object  of  these  experiments  has  been  to  throw  some  light  on  the 
problem  as  to  what  organisms  are  most  likely  to  effect  such  a  decom- 
position, and  whether  there  is  an  appreciable  production  of  sugar  as 
a  result  of  bacterial  activity.  The  four  classes  of  hemicelluloses  under 
special  investigation  have  been  represented  by  the  following  sub- 
stances: 

Pentosans  Dulse.  Mannans  Salep. 

~  ,  J  Irish  Moss.  Levulans  Sinistrin. 

Galactans  <  _  . 

[  Limu  Manauea. 


324 


Mary  Davies  Swartz, 


Both  aerobic  and  anaerobic  cultures  have  been  made,  in  neutral, 
faintly  alkaline,  and  faintly  acid  reaction,  with  solutions  made  from 
the  carbohydrates  alone,  and  with  the  addition  of  small  amounts  of 
such  nutrients  as  beef  extract  or  peptone  to  facilitate  the  growth  of 
the  organisms. 

Anaerobic  cultures  in  test  tubes  have  been  made  by  the  Wright 
method ;  anaerobic  cultures  in  Erlenmeyer  flasks,  by  passing  a  stream 
of  hydrogen  through  for  half  an  hour,  and  then  sealing  hermetically. 

The  aerobes  which  have  been  employed  all  occur  in  the  human 
digestive  tract.  Both  aerobic  and  anaerobic  cultures  from  the 
faeces  of  human  subjects  have  also  been  used,  in  conjunction  with 
soil  bacteria  from  street  sweepings. 

Tests  for  the  presence  of  reducing  sugar  have  been  made  by  pre- 
cipitating the  carbohydrates  in  solution  with  absolute  alcohol,  evapor- 
ating the  alcoholic  extract  to  dryness,  taking  up  the  residue  in  2  or 
3  cc.  of  water,  and  boiling  two  minutes  with  Fehling's  solution. 

Suitable  controls  have  been  used  in  all  cases. 

TRIALS  WITH  PURE  CULTURES  OF  AEROBES. 

One  per  cent  solutions  of  the  preparations  from  dulse,  Irish  moss  and 
salep,  neutral,  acid,  and  alkaline  in  reaction,  and  consisting  of,  (1) 
pure  carbohydrate:  (2)  carbohydrate  plus  J  per  cent  beef  extract  and 
\  per  cent  sodium  chloride;  (3)  carbohydrate  plus  1  per  cent  peptone 
and  i  per  cent  sodium  chloride,  have  been  used  as  culture  media. 
Five  cc.  portions  of  each  of  these  solutions  were  placed  in  test-tubes 
with  a  pipette,  and  inoculated  with  the  following  organisms:  B.  Coli 
communis,  B.  Pyocyaneus,  B.  Prodigiosus,  B.  Proteus  vulgaris, 
B.  Pyogenes  foetidus. 

To  approximate  the  conditions  in  ordinary  digestion  of  these  car- 
bohydrates, they  were  incubated  for  three  days  at  a  temperature  of 
37.5°  C.  At  the  end  of  this  time,  nearly  all  gave  evidence  of  some 
bacterial  growth.  Salep-peptone  cultures  of  B.  Pyocyaneus  showed 
a  brilliant  green;  salep  solutions  containing  B.  Pyogenes  foetidus, 
and  B.  Coli  in  alkaline-beef  extract  media,  had  changed  from  trans- 
parent colorless  solutions  to  an  opaque  white  jelly  insoluble  in  water. 

The  carbohydrates  were  then  precipitated  with  alcohol,  and  after 
standing  several  days  were  compared  with  controls  similarly  prepared, 
to  see  whether  any  change  could  be  observed  in  the  nature  or  amount 
of  carbohydrate.  The  results  were  in  all  cases  negative.  These  pre- 
cipitates were  then  transferred  to  small  folded  filter  papers  of  uniform 


Nutrition  Investigations. 


325 


weight,  previously  prepared.  The  alcoholic  filtrates  were  tested  for 
sugar;  the  precipitates  were  dried,  and  their  weight  compared  with 
that  of  the  control.  It  was  thought  that  this  rather  crude  method 
would  show  whether  any  considerable  amount  of  the  carbohydrate 
had  disappeared.  The  results  were  so  largely  negative  that  weighings 
of  every  precipitate  were  not  made.  There  seemed  to  be  a  slight  loss 
of  dulse,  in  some  of  the  cultures  of  B.  Proteus  vulgaris,  B.  Pyogenes 
foetidus,  and  B.  Coli  communis,  but  repetition  of  these  experiments 
allowing  the  organisms  in  question  to  grow  two  weeks,  not  only  in 
dulse  but  also  in  salep  media,  did  not  justify  any  conclusion  that  an 
appreciable  amount  of  carbohydrate  had  disappeared. 
All  tests  for  reducing  sugar  were  negative. 

Four  per  cent  solutions  of  Irish  moss,  and  two  per  cent  solutions 
of  limu  manauea  were  then  prepared,  with  reactions  and  additions  of 
nutrient  material  as  described  in  the  first  series  of  experiments.  These 
formed  firm  jellies,  which  were  used  to  study  the  possibility  of  lique- 
faction or  gas  formation.  Stab  cultures  were  made,  and  grown  at  a 
temperature  of  25°-30°  C.  for  one  to  three  weeks.  No  liquefaction 
or  gas  formation  was  observed  in  any  case. 

TRIALS  WITH  MIXTURES  OF  AEROBES. 

Mixtures  of  B.  Pyocyaneus,  B.  Prodigiosus,  B.  Proteus  vulgaris, 
and  B.  Pyogenes  foetidus,  were  used,  also  mixtures  of  faecal  and  soil 
bacteria.  These  were  first  inoculated  into  nutrient  bouillon,  the 
former  from  pure  cultures,  the  latter  from  human  faeces  and  street 
sweepings,  and  incubated  24  hours.  Five  cc.  portions  of  these  cultures 
were  then  introduced  into  50  cc.  of  neutral  solutions  of  each  of  the 
different  carbohydrates,  in  small  Erlenmeyer  flasks,  and  these  cul- 
tures allowed  to  grow  for  four  weeks  at  37.5°  C.  At  the  end  of  this 
time,  no  marked  change  had  taken  place  save  in  the  salep  culture  of 
B.  Pyocyaneus,  B.  Proteus  vulgaris,  B.  Pyogenes  foetidus  and  B. 
Prodigiosus.  This  had  changed  from  a  colorless,  semi-transparent, 
slightly  mucilaginous  fluid,  to  a  firm,  white  opaque  jelly,  insoluble 
in  water,  but  readily  soluble  in  dilute  alkali;  a  phenomenon  already 
observed  with  this  carbohydrate  in  cultures  of  B.  Coli  communis  and 
B.  Pyogenes  foetidus.  No  liquefaction  had  taken  place  with  Irish 
moss  nor  limu  manauea. 

The  carbohydrates  were  then  precipitated  with  alcohol,  the  alco- 
holic extracts  tested  for  sugar,  and  the  precipitates  hydrolyzed  by 
boiling  with  2  per  cent  hydrochloric  acid,  neutralized,  made  up  to  a 


326 


Mary  Davies  Swartz, 


definite  volume,  and  examined  in  a  polariscope.  The  results  of  these 
experiments  are  shown  in  the  following  table.  Mixtures  of  B.  Pyo- 
cyaneus,  B.  Prodigiosus,  B.  Proteus  vulgaris  and  B.  Pyogenes  foetidus 
are  designated  A,  and  mixtures  of  faecal  and  soil  bacteria,  B. 


REDUCTION 

ROTATION  AFTER  HYDROLYSIS. 

SUBSTANCE 

BACTERIAL 

OF 

CULTURE 

fehling's 
solution. 

Experiment. 

Control. 

Dulse  

B 

+  0.13° 

+0.20° 

A 

+0.20° 

B 

+0.27° 

+0.20° 

Limu  Manauea  .... 

A 

Not  determined. 

Salep  

A 

Not  determined. 

Salep  

B 

+0.17° 

+0.20° 

Sinistrin  

A 

-0.97° 

—0.97° 

The  action  of  putrefactive  organisms  upon  the  dulse  preparation 
was  also  studied,  according  to  the  method  used  by  Slowtzoff  (154) 
in  the  case  of  xylan.  One  hundred  grams  of  chopped  lean  beef  and 
10  grams  of  sodium  carbonate  were  added  to  1  liter  of  water,  and 
the  mixture  allowed  to  stand  in  a  warm  place  for  three  days.  Two 
hundred  and  fifty  cc.  were  then  removed  for  a  control,  and  to  the 
remainder  0.5  gram  of  dulse  was  added.  This  solution  gave  a  strong 
pentosan  reaction;  the  control  was  pentosan-free.  The  two  solutions 
were  put  in  a  warm  place,  and  tested  daily  for  pentosans.  After  five 
days'  digestion,  the  reaction  of  the  dulse  solution  was  very  much 
fainter  than  at  first,  but  it  did  not  entirely  disappear  till  the  twelfth 
or  thirteenth  day.  Slowtzoff  found  that  xylan  disappeared  in  nine 
or  ten  days,  but  his  solution  was  kept  at  a  temperature  of  40°  C, 
while  these  mixtures  remained  at  a  temperature  of  from  30°  to  35°  C, 
a  condition  less  favorable  for  rapid  decomposition. 

Solutions  of  Irish  moss  were  digested  with  faecal  mixtures  in  the 
following  manner:  Human  faeces  were  rubbed  to  a  mud  with  water. 
Ten  cc.  portions  of  this  material  were  added  to  flasks  containing  50  cc. 
of  a  1  per  cent  "moss"  solution,  and  allowed  to  digest  in  a  warm  place 
for  24  hours.  A  portion  of  water  inoculated  in  the  same  way  was 
used  as  a  control.  Small  portions  of  these  solutions  were  then  evap- 
orated nearly  to  dryness,  extracted  with  alcohol,  and  tested  for  reduc- 
ing sugar.    The  results  were  wholly  negative. 

That  limu  manauea  is  not  entirely  resistant  to  the  action  of  putre- 
fying organisms  is  shown  by  the  following:  A  solution  was  made 


Nutrition  Investigations. 


327 


up  to  contain  2  per  cent  of  the  air  dry  extract,  1  per  cent  peptone, 
\  per  cent  beef  extract  and  \  per  cent  sodium  chloride.  This  could  be 
filtered  through  paper  only  on  a  hot,  water-jacketed  funnel,  from 
which  it  dropped  as  a  clear,  amber-colored  jelly.  After  standing 
unsterilized  over  night  in  a  warm  room,  this  was  found  to  be  entirely 
broken  up  by  the  formation  of  gas  throughout  the  whole  mass.  The 
reaction,  which  had  been  neutral,  was  now  acid  to  litmus.  This 
material  was  placed  in  a  flask  and  allowed  to  stand  for  two  months, 
at  the  end  of  which  time,  the  greater  portion  was  liquefied,  the  former 
lumps  of  jelly  being  reduced  to  small  particles  distributed  throughout 
the  liquefied  portion.  Alcoholic  extracts  did  not  reduce  Fehling's 
solution.  A  sterile  preparation  of  the  plain  manauea  extract  in  test 
tubes  was  inoculated  with  some  of  this  material,  but  without  produc- 
ing the  same  striking  results.  There  were  evidences  of  growth,  but 
none  of  liquefaction  or  gas  formation,  in  the  course  of  two  weeks. 

TRIALS  WITH  ANAEROBES. 

The  action  upon  Irish  moss  of  pure  cultures  of  the  powerful  putre- 
factive organisms  B.  Putrificus,  Bienstock,  B.  Maligni  cedematis, 
and  B.  Anthracis  symptomatici,  was  tried  in  the  following  way.  A 
4  per  cent  solution  of  the  moss  was  prepared,  which  would  not  become 
liquefied  at  a  temperature  of  30°-35°  C.  From  this  material  culture 
media  were  prepared,  neutral,  alkaline,  and  acid  in  reaction,  using 
the  solution  plain,  and  with  the  addition  of  \  per  cent  beef  extract 
and  \  per  cent  salt,  or  1  per  cent  peptone  and  \  per  cent  salt.  Test 
tubes  were  inoculated  from  fresh,  active  cultures,  and  the  organisms 
allowed  to  grow  for  one  to  three  weeks,  being  examined  at  first  daily, 
and  later  every  three  or  four  days,  for  liquefaction  and  gas  formation. 
The  results  were  negative  in  all  cases,  save  that  in  the  peptone  media 
an  occasional  small  bubble  was  seen,  with  cultures  of  the  bacilli  of 
malignant  cedema  and  symptomatic  anthrax.  However,  the  same 
phenomena  were  observed  in  peptone-agar  tubes  used  as  controls. 

Mixtures  of  B.  Anthracis  symptomatici  and  B.  Maligni  cedematis 
were  tried  upon  solutions  of  dulse,  Irish  moss,  salep  and  sinistrin,  in 
the  following  way:  Small  Erlenmeyer  flasks  containing  50  cc.  of 
1  per  cent  solutions  of  each  of  these  carbohydrates,  and  5  cc.  of  ordi- 
nary nutrient  bouillon,  were  inoculated  with  fresh  cultures  of  these 
organisms,  rendered  anaerobic,  and  incubated  for  four  weeks  at  37.5° 
C.  On  inspection,  no  change  was  apparent.  The  carbohydrates 
were  removed,  the  alcoholic  extracts  examined  for  reducing  sugar,  and 


328 


Mary  Davies  Swartz, 


the  carbohydrate  residues  hydrolyzed  and  examined  in  the  polari- 
scope,  as  in  similar  trials  with  aerobes.  The  results  are  shown  in 
the  following  table: 


REDUCTION  OF 

ROTATION  AFTER  HYDROLYSIS. 

NAME   OF  SUBSTANCE. 

fehling's 

SOLUTION. 

Experiment. 

Control. 

Dulse  

Lost  by 

accident 

Irish  Moss  

+  0.24° 

+  0.20° 

Salep  

+ 

+  0.13° 

+  0.20° 

Sinistrin  

+ 

-  0.27° 

-  0.97° 

Mixtures  of  soil  and  faecal  bacteria  were  also  tried,  the  experiments 
being  carried  out  just  as  described  for  mixtures  of  the  bacilli  of  symp- 
tomatic anthrax  and  malignant  cedema.  The  results  are  shown  in 
the  following  table: 


NAME  OF  SUBSTANCE. 


reduction  of 
febxing's 
solution. 


ROTATION  AFTER  HYDROLYSIS. 


Experiment. 


Control. 


Dulse  

Irish  Moss 
Salep  


+ 


+  0.13° 
+  0.20° 
+  0.03° 


+  0.20° 
+  0.20° 
+  0.20° 


DISCUSSION  AND  SUMMARY. 


It  seems  reasonable  to  expect,  that  if  the  hemicelluloses  used  in 
these  trials  were  readily  attacked  by  micro-organisms,  there  would  have 
been  some  evidence  of  change  in  three  days,  if  conditions  for  growth 
were  favorable  as  regards  reaction  and  temperature;  but  although  the 
concentration  of  the  solutions  was  moderate,  the  reaction  varied, 
and  temperature  37.5°  C,  results  were  negative,  even  in  the  cases 
where  nutrients  were  added  to  facilitate  bacterial  growth.  Apparently 
all  of  the  material  was  recovered  in  unaltered  condition,  save  in 
certain  instances  where  salep  underwent  an  insoluble  modification. 

In  trials  where  the  cultures  were  allowed  to  grow  from  one  to  three 
weeks,  no  difference  in  the  results  could  be  detected,  by  the  methods 
employed.  In  solid  media  there  was  no  liquefaction  and  practically 
no  gas  formation,  except  in  the  case  of  the  peptone-beef  extract 
preparation  of  limu  manauea,  on  exposure  to  the  air. 


Nutr  it  ion  Investigations . 


329 


Marked  evidences  of  change  were  observed  in  one  trial  with  a  putre- 
factive mixture  (on  dulse),  and  in  some  of  the  four-week  cultures. 

Irish  moss  was  the  most  thoroughly  investigated  and  proved  the 
most  resistant.  In  the  long  experiments  (4  weeks)  where  the  other 
carbohydrates  suffered  more  or  less  change  this  one  remained  appar- 
ently unaltered.  The  results  of  this  series  are  summarized  in  the 
following  table: 

Irish  Moss. 


CULTURES  USED. 


Mixture  of  Pure  Aerobes 


reduction  of 
fehling's 
solution. 


ROTATION  OF  UNALTERED  CAR- 
BOHYDRATE AFTER  HYDROLYSIS. 


Irish  Moss. 

Control. 

+  0.20° 

+  0.20° 

+  0.27° 

+  0.20° 

+  0.24° 

+  0.20° 

+  0.20° 

+  0.20° 

Mixture  of  Faecal  and  Soil  Bacteria 
(aerobic)  

Mixture     of     Bacilli    of  Malignant 
Oedema  and  Symptomatic  Anthrax .  . 

Mixture  of  Faecal  and  Soil  Bacteria 
(anaerobic)  


The  single  experiment  with  the  galactan,  limu  manauea,  under  the 
same  conditions,  with  the  mixture  of  pure  aerobes,  gave  similar 
results,  but  the  fact  that  liquefaction  occurred  in  the  peptone-beef 
extract  culture  medium  after  exposure  to  the  air,  shows  that  general 
conclusions  as  to  the  behavior  of  galactans  cannot  be  drawn  from 
study  of  a  single  representation  of  the  class.  We  have,  however, 
further  proof  that  the  galactans  are  not  easily  decomposed  by  bacteria, 
in  the  fact  that  aqueous  solutions  of  all  the  galactans  included  in  the 
present  series,  could  be  left  several  days  in  the  warm  atmosphere  of 
the  laboratory  without  any  apparent  change  taking  place;  and  in  the 
fact  that  agar-agar,  so  widely  used  in  bacteriological  laboratories 
on  account  of  its  indifference  to  bacterial  action,  is  a  member  of  the 
galactan  group.  It  has  been  suggested1  that  extracts  of  other  sea- 
weeds might  prove  good  substitutes  for  agar-agar  as  culture  media, 
if  fully  investigated.  So  far,  the  greatest  objection  to  use  of  Irish 
moss  in  this  way  is  that  it  tends  to  liquefy  at  body  temperature; 
strong  solutions  (4  per  cent)  can,  however,  be  kept  fairly  firm  at  a 


xCf.  Reed  (18). 


330 


Mary  Davies  Swartz, 


temperature  of  30°  C.  The  extract  of  limu  manauea  is  free  from 
these  objections,  but  extensive  experiment  is  still  necessary  to  demon- 
strate its  powers  of  resistance. 

The  soluble  dulse  pentosan  is  certainly  decomposed  not  only  by 
putrefactive  organisms  under  the  most  favorable  conditions  (e.g.,  in 
meat  mixtures),  but  by  aerobes  and  anaerobes  in  solutions  where  the 
carbohydrate  is  the  chief  source  of  nutriment.  The  results  of  the 
four  weeks'  digestions  are  summarized  in  the  following  table: 


Dulse. 


CULTURES  USED 

REDUCTION 
OP 

fehling's 
solution. 

ROTATION  OF  UNALTERED 
CARBONYDRATE  AFTER 
HYDROLYSIS. 

Dulse. 

Control. 

Mixture  of  Faecal  and  Soil  Bacteria 
(aerobic)  

+0.13° 

(Lost  by 
Accident) 

+0.13° 

+0.20° 
+0.20° 

+0.20° 

Mixture  of  the  Bacilli  of  Malignant 
Oedema  and  Symptomatic  Anthrax.  . 

Mixture  of  faecal  and  Soil  Bacteria 

In  the  present  studies,  this  pentosan  stands  second  to  the  galactans 
in  degree  of  resistance. 

Sawamura  (267)  thought  that  he  observed  a  slight  hydrolysis  o: 
mannan  by  B.  Prodigiosus,  an  observation  which  has  not  been  verifiec 
in  these  experiments.  No  reducing  substance  was  detected  in  the 
three-day  cultures  nor  the  four-weeks  cultures,  in  which  this  organism 
was  present.  The  opaque  jelly,  insoluble  in  water,  formed  from  salep 
by  the  action  of  B.  Coli  communis,  B.  Prodigiosus,  and  mixed  cultures 
containing  these  organisms,  resembles  an  intermediary  product  o: 
the  acid  hydrolysis  of  salep-mannan  described  by  Thamm  (276) 
He  isolated  and  examined  two  such  products,  one  forming  an  opales- 
cent solution  in  water,  the  other  insoluble,  but  passing  over  into  the 
soluble  form  by  treatment  with  dilute  alkali;  both  were  anhydrides 
of  mannose.  It  seems  reasonable  to  inquire  whether  this  insoluble 
material  produced  by  bacterial  action  may  not  be  regarded  as  an 
early  stage  in  the  hydrolysis  of  the  carbohydrate  under  consideration 
especially  in  view  of  the  fact  that  in  all  the  other  four-week  trials  a 
very  definite  reduction  of  Fehling's  solution  was  noted,  corresponding 


Nutrition  Investigal it ms. 


331 


in  strength  with  the  loss  of  unaltered  carbohydrate,  as  shown  in  the 
following  summary: 

Salep. 


CULTURES  USED. 


ROTATION  OF  UNALTERED  CARBO- 
REDUCTION  OF   j     HYDRATE  AFTER  HYDROLYSIS. 

FEHLING  'S   

SOLUTION. 


Salep. 


Control. 


Mixture  of  Pure  Aerobes . 


Mixture  of  Faecal  and  Soil  Bacteria 
(aerobic)  


(Insoluble 
jelly) 

+ 


Mixture  of  the  Bacilli  of  Malignant 
Oedema  and  Symptomatic  Anthrax. . 

Mixture  of  Faecal  and  Soil  Bacteria 
(anaerobic)  


Not  det  ermined 


+  0.17°  +0.20° 


+  0.13°  +0.20° 


+  0.03°  +0.20° 


These  experiments  give  some  grounds  for  expecting  the  hydrolysis 
of  salep  in  the  alimentary  tract,  through  the  action  of  bacteria. 
Two  experiments  with  sinistrin  gave  the  following  results: 


Sinistrin. 


ROTATION  OF  UNALTERED  CAR- 

REDUCTION OF 

BOHYDRATE  AFTER  HYDROLYSIS. 

CULTURES  USED. 

FEHLING 'S 

SOLUTION. 

Sinistrin. 

Control. 

Mixture  of  Pure  Aerobes  

-  0.97° 

-0.97° 

Mixture  of  Bacilli  of  Malignant  Oedema 

and  Symptomatic  Anthrax  

+ 

-  0.27° 

-  0.97° 

Sinistrin  is  therefore  hydrolyzed  by  the  anaerobic  putrefactive 
organisms,  but  further  experiments  are  necessary  to  determine  how 
readily  this  change  takes  place. 


Physiological  Investigations. 


In  the  physiological  experiments,  attempts  have  been  made  to 
answer  the  following  questions:  (1)  To  what  extent  are  hemicelluloses 
digested  by  animal  and  vegetable  enzymes?  (2)  Can  they  be  ab- 
sorbed and  utilized  without  intervention  of  the  alimentary  tract? 


332 


Mary  Dames  Swartz, 


(3)  Do  they  reappear  in  the  faeces  after  administration  per  os?  The 
various  experiments  will  accordingly  be  discussed  in  these  three 
groups:  (1)  Trials  with  Enzymes;  (2)  Parenteral  Trials;  (3)  Feeding 
Experiments. 

TRIALS  WITH  ENZYMES. 

Approximately  1  per  cent  solutions  of  the  various  hemicelluloses 
(with  the  exception  of  Limu  Lipoa,  which  was  finely  ground  and  sus- 
pended in  water),  have  been  digested  for  24  hours  at  37.5°  C.  in  the 
presence  of  toluene,  with  the  following  enzymes:  (1)  Filtered  human 
saliva.  (2)  Malt  diastase,  dialyzed  sugar-free.  (3)  "Taka"  dias- 
tase (Eurotium  oryzae).  (4)  Chloroform  extract  of  pig's  pancreas. 
(5)  Fresh  pancreatic  juice  of  dogs.  (6)  Chloroform  water  extract 
of  dog's  intestines.    (7)  Glycerol  extract  of  pig's  stomach. 

Digestions  have  also  been  made  with  0.2  per  cent  hydrochloric  acid, 
to  determine  whether  any  of  the  action  of  the  artificial  gastric  juice 
might  be  due  to  the  acid  present.  The  activity  of  the  amylolytic 
enzymes  has  always  been  tested  first  with  starch  paste,  and  that  of 
the  gastric  extract  with  fibrin.  Boiled  controls  have  been  employed 
in  every  instance,  and  all  trials  have  been  made  in  duplicate. 

Tests  for  reducing  sugar  have  been  conducted  in  the  following 
manner:  At  the  end  of  24  hours  the  solutions  were  evaporated  to 
thick  syrups  on  the  water  bath,  to  free  from  toluene  and  to  concen- 
trate so  that  the  undigested  hemicelluloses  could  be  readily  precipi- 
tated by  absolute  alcohol.  The  alcoholic  extracts  were  filtered  off 
and  evaporated  to  dryness;  the  residues  were  taken  up  in  a  few  drops 
of  water  and  tested  for  sugar  with  Fehling's  solution.  The  results 
of  all  digestion  trials  are  shown  in  the  table  on  opposite  page. 

PARENTERAL  INJECTIONS. 

Methods  and  Technique. 

Small  dogs  were  used  for  all  injections,  after  a  confinement  in  cages 
long  enough  to  obtain  samples  of  normal  urine.  The  carbohydrates 
employed  in  these  experiments  were  preparations  of  dulse,1  Irish  moss,2 
salep,3  and  sinistrin.4   They  were  introduced  subcutaneously ,  by  means 


iCf.  p.  303. 

2Cf.  p.  308. 
3Cf.  p.  312. 
<Cf.  p.  315. 


0.2  PER  CENT. 

HCl. 

I  I  I  I  +  I  ++  I 

pM  0 

+ 

GASTRIC 
EXTRACT. 

III         +        +         |  + 

INTESTINAL 
EXTRACT. 

1      1      1      1      1      1      1      1  1 

PANCREATIC 
JUICE. 

1      1      1      1      1      1      1      1      1  1 

PANCREATIC 
EXTRACT. 

1           1  1 

"taka" 
diastase. 

+  I  I  I  1  I  \  +  f  .|S  + 

+ 

MALT  DIASTASE. 

1    1    1    1    1    1    1    1    1    1  1 

SALIVA. 

1    1    1    1    1    1    1    1    1    1  1 

SOURCE. 

Dulse 

Limu  Lipoa 
Irish  Moss 
Limu  Manauea 
Limu  Huna 
Lirrm  Akiaki 

Limu  Uaualoli 
Limu  Kohu 
Slippery  Elm 
Salep 

Sinistrin 

CLASS. 

Pentosan  

Mannan  

334 


Mary  Davies  Swartz, 


of  a  syringe,  or  intraperitoneally,  by  means  of  a  needle  and  burette 
with  pressure-bulb  attached,  always  under  aseptic  conditions.  After 
receiving  injections,  the  animals  were  replaced  in  cages,  and  the 
urine  collected  under  toluene.  The  excess  of  toluene  was  removed, 
at  the  time  of  examination,  by  means  of  a  separatory  funnel,  and  the 
urine  measured,  filtered,  and  tested  for  reducing  substances  with 
Fehling's  solution. 

Qualitative  tests  for  the  carbohydrates  were  made  in  the  following 
manner:  (l)  for  dulse  and  salep,  by  boiling  a  few  drops  of  urine  with 
Fehling's  solution,  from  which  these  hemicelluloses  were  precipitated 
in  fine  white  flocks,  even  if  only  traces  were  present;  (2)  for  Irish 
moss,  by  the  reduction  of  Fehling's  solution  after  hydrolysis  of  the 
urine  with  dilute  hydrochloric  acid;1  (3)  for  sinistrin,  by  the  marked 
increase  in  the  levo-rotation  of  the  urine. 

Isolation  of  the  carbohydrates  was  accomplished  by  freeing  the 
urine  from  inorganic  salts  with  lead  acetate,  removing  the  excess  of 
lead  with  hydrogen  sulphide,  and  concentrating  the  salt-free  solutions 
to  a  small  volume.  Dulse  and  Irish  moss  were  then  precipitated  with 
absolute  alcohol;  salep  with  alcohol  or  Fehling's  solution;  sinistrin 
with  milk  of  lime,  being  freed  from  its  calcium  compound  by  the 
method  used  in  its  preparation.2 

These  substances  were  identified  as  carbohydrates,  by  their  yield- 
ing reducing  sugar  on  hydrolysis;  salep  and  sinistrin  were  further 
identified  by  their  levo-rotation,  Irish  moss  by  testing  for  mucic  acid, 
and  dulse  by  testing  for  furfurol. 

Quantitative  determinations  of  dulse,  salep  and  sinistrin  were  made 
by  polariscopic  examination  in  a  200  mm.  tube,  all  samples  of  urine 
being  clarified  with  equal  volumes  of  alumina  cream.  A  satisfactory 
quantitative  method  for  the  determination  of  Irish  moss  was  not 
developed.  It  proved  impossible  to  estimate  any  of  these  carbohy- 
drates quantitatively  by  the  method  of  acid  hydrolysis.  In  some 
instances,  especially  with  Irish  moss,  a  trace  of  reduction  was  ob- 
tained, but  in  most  cases,  the  results  were  negative,  although  the  hemi- 
cellulose  was  known  to  be  present.3 


^rial  was  made  of  Bauer's  method  (Zeitschrift  fiir  physiologische  Chemie,  51, 
p.  158,  1907)  of  determining  galactose  in  urine  as  mucic  acid,  by  concentrating 
100  cc.  of  urine  with  25-35  cc.  of  concentrated  nitric  acid  (sp.  gr.  1.4)  to  a  volume  of 
20  cc,  but  owing  probably  to  the  low  percentage  of  galactose  from  the  small  amount 
of  Irish  moss  present,  this  test  was  unsatisfactory. 

2Cf.  p.  315. 

3Samples  were  removed  and  tested  every  half  hour  for  2\  hours.  At  the  end  of 
1  hour  they  were  usually  neutral,  or  slightly  alkaline  in  reaction.    Addition  of  suf- 


Nutrition  Investigations . 


335 


INJECTIONS  OF  DULSE. 

L  Subcutaneous. 

A  dog  weighing  11  kg.  received  60  cc.  of  a  dulse  solution  contain- 
ing 0.9  grams  of  pure  substance.  No  reduction  of  Fehling's  solution 
was  observed  at  any  time.  The  time  and  rate  of  dulse  excretion  are 
shown  in  the  following  table : 

Examination  of  Urine. 


ESTIMATED  EXCRETION 
OF  DULSE.  * 


cc. 

Grams. 

February  1,  12:30  P.M  

-0.14°f 

February  1,   1  P.M  

Injection 

February  2,  10  A.M  

226 

-0.62° 

0.61 

February  3,  10  A.M  

250 

-0.55° 

0.57 

February  4,  10  A.M  

150 

-  0.41° 

0.21 

February  5,  10  A.M  

210 

-  0.34° 

0.21 

February  6,  10  A.M  

310 

-  0.28° 

0.04 

February  7,  10  A.M  

-  0.20° 

Total  

1.64 

2.  Intraperitoneal. 

The  same  dog  received  in  this  experiment  75.6  cc.  of  a  dulse  solu- 
tion containing  1.4  grams  of  pure  substance.  No  reduction  of  Feh- 
ling's solution  was  observed  before  or  after  the  injection.  The  time 
and  rate  of  dulse  excretion  are  shown  in  the  following  table: 

Examination  of  Urine. 


ROTATION. 


ESTIMATED  EXCRETION 
OF  DULSE.* 


December  3,  2  P.M 
December  3,  3  P.M 
December  4,  10  A.M 
December  5,  10  A.M 
December  5,  12  M .  . 
December  6  and  7. . 
December  8, 10  A.M 
December  9,  10  A.M 


Injection 
133 
200 
115 
383 
520 
350 


-0.14°t 

-  0.62° 

-  0.52° 

-  0.28° 

-  0.48° 

-  0.28 
-0.20 


Grams. 


0.36 
0.42 
0.05 
0.69 
0.24 


Total. 


1.76 


ficient  hydrochloric  acid  to  make  the  strength  2  per  cent  caused  no  subsequent 
production  of  sugar. 

*  All  readings  have  been  taken  on  the  Ventzke  scale,  and  calculated  as  angular  degrees. 

t  Estimating  normal  rotation  of  urine  as  — 0.17°  (average). 


336 


Mary  Davies  Swartz, 


In  both  these  experiments,  the  presence  of  dulse  was  readily  detected 
by  Fehling's  solution  in  every  urine  which  showed  a  high  rotation. 
From  the  samples  of  the  first  48  hours  after  injection,  a  considerable 
amount  was  isolated  and  identified  as  carbohydrate.  It  is  evident 
that  the  excretion  of  this  pentose-carbohydrate  is  gradual,  commenc- 
ing soon  after  the  injection,  and  continuing  from  four  to  five  days. 
While  any  quantitative  estimate  of  the  amount  excreted,  based  on  the 
changes  in  rotation,  is  subject  to  a  high  percentage  of  error,  owing  to 
normal  fluctations  in  the  rotation  of  the  urine,  as  well  as  to  analyt- 
ical discrepancies  unavoidable  in  dealing  with  solutions  containing 
only  minute  quantities  of  the  substance  under  investigation,  it  is  evi- 
dent that  most  of  the  dulse  must  have  been  excreted,  and  that,  too, 
without  any  essential  change  in  character. 

INJECTIONS  OF  IRISH  MOSS. 

1.  Subcutaneous. 

A  dog  weighing  9.4  kg.  received  100  cc.  of  Irish  moss  solution,  con- 
taining 1.5  grams  of  dry  substance.  No  reducing  substance  occurred 
in  the  urine.  Changes  in  rotation,  due  to  the  injection,  are  shown  in 
the  following  table: 


Examination  of  Urine. 


TIME. 

VOLUME. 

ROTATION. 

IRISH  MOSS. 

May  18,  9  A.M  

CC. 

-0.04° 

May  18,  4  P.M  

Injection 

May  19,  9  A.M  

128 

+  0.34° 

May  20,  9  A.M  

226 

+  0.06° 

May  21,  11  A.M  

330 

-0.20° 

May  22,    9  A.M  

370 

-0.14° 

Tests  for  Irish  moss  on  May  19th  were  negative,  but  on  May  20th- 
22nd  they  were  faintly  positive.  The  experiment  was  discontinued 
at  this  point.  The  injection  was  not  very  well  borne,  the  dog  remain- 
ing lethargic  throughout  the  period. 

2.  Intraperitoneal. 

Experiment  A .  A  dog  weighing  10  kg.  received  160  cc.  of  an  Irish 
moss  solution  containing  1.3  grams  air  dry  material.  Examination 


Nutrition  Investigations. 


337 


for  the  presence  of  carbohydrate  was  made  by  testing  the  urine  for 
reducing  substances,  before  and  after  hydrolysis.  The  results  are 
shown  in  the  following  table: 

Examination  of  Urine. 


REDUCTION   OF   FEHLING  S 
SOLUTION. 


Before 
Hydrolysis. 


After  Hydroly- 


October  13,  11  A.M  

October  13,  12 :30  P.  M  |  Injection 

October  13,  2  P.M  ,  ..  27 

October  13,  5  P.M   60 

October  14,  9  A.M   450 

October  15,  5  P.M   45 

October  16,  9:30  A.M  


The  urine  before  the  injection  showed  a  rotation  of  —0.14°,  a 
sample  of  the  mixed  urines  of  October  13,  5  P.M.,  and  October  14, 
9  A.M.,  showed  a  rotation  of  —0.034°,  the  diminished  levo-rota- 
tion  undoubtedly  due  to  the  presence  of  this  dextro-rotatory  carbohy- 
drate. On  hydrolysis,  50  cc.  of  this  mixed  sample  yielded  sugar 
equivalent  to  0.035  grams  of  dextrose  (by  Allihn's  method).  From  the 
remainder  of  this  sample,  Irish  moss  carbohydrate  was  isolated;  it 
formed  a  grayish-white  powder,  swelling  in  water,  and  yielding  mucic 
acid  on  oxidation  with  nitric  acid. 

Experiment  B.  A  dog  weighing  9  kg.  received  intraperitoneally 
100  cc.  of  a  2  per  cent  solution  of  Irish  moss  preparation.  Examina- 
tion for  carbohydrate  was  made  as  in  the  preceding  experiments. 
The  results  appear  in  the  following  table: 


Examination  of  Urine. 


REDUCTION  OF  FEHLING 'S 

SOLUTION. 

TIME. 

VOLUME. 

Before  Hydroly- 

After Hydroly- 

sis. 

sis. 

CC. 

October  30,  1  P.M  

October  30,2:30  P.M  

Injection 

October  31,    9  A.M  

250 

November  1, 10  A.M  

200 

+ 

November  2,  10  A.M  

115 

Irish  moss  was  isolated  and  identified  in  the  urine  of  November  1st. 


338 


Mary  Davies  Swartz, 


INJECTIONS  OF  SALEP. 

i.  Subcutaneous. 

A  dog  weighing  7.2  kg.  received  56  cc.  of  salep  solution,  containing 
0.75  grams  of  pure  mannan.  No  reducing  substance  was  found  in 
the  urine.  The  changes  in  rotation,  due  to  salep,  are  shown  in  the 
following  table : 


Examination  of  Urine. 


TIME. 

VOLUME. 

ROTATION. 

ESTIMATION   OF  AMOUNT 
OF  SALEP  EXCRETED. 

CC. 

Grams. 

May  17,  

-  0.17° 

May  18,3:30P.M  

Injection 

May  19,9  A.M  

May  20,9  A.M  

138 

-  0.27° 

0.3 

May  21,  9  A.M  

132 

-  0.27° 

0.3 

May  22,9  A.M  

114 

-  0.20° 

0.04 

May  22,  5  P.M  

127 

-  0.14° 

Salep  was  isolated  and  identified  in  the  urines  of  May  20,  21,  and  22. 


2.  Intraperitoneal. 

Experiment  A .  A  dog  weighing  7  kg.  received  68  cc.  of  salep  solu- 
tion, containing  1.2  grams  of  air  dry  mannan.  No  reducing  substance 
was  present  in  the  urine  at  any  time.  Tests  for  the  presence  of  salep 
by  means  of  Fehling's  solution,  gave  the  following  results: 


Examination  of  Urine. 


TIME. 

VOLUME. 

SALEP  PRESENT. 

October  21,  12  M  

October  21,  2:30  P.M  

October  22,  9  A.M  

October  23,  9  A.M  

October  24,  9  A.M  

CC. 

Injection 
125 
190 
140 

+ 
+ 

The  salep  was  easily  isolated  and  identified  in  the  urine  of  October 
22  and  23,  the  sugar  obtained  on  hydrolysis  being  equivalent  to  0.33 
grams  salep. 

Experiment  B.    A  dog  weighing  9.2  kg.  received  80  cc.  of  salep  so- 


Nutrition  Investigations. 


339 


lution,  containing  1.4  grams  of  air  dry  substance.  No  reducing  sub- 
stance was  detected  in  any  of  the  urines.  Tests  for  salep  with  Feh- 
ling's solution  gave  the  following  results: 


Examination  of  Urine. 


TIME. 

VOLUME. 

SALEP  PRE8ENT. 

October  24,  11  A.M  

October  24, 12  M  

October  25, 12  M  

October  26, 10  A.M  

October  27, 10  A.M  

October  28, 10  A.M  

CC. 

Injection 
155 
180 
180 

+ 
+ 
+ 

From  the  urine  of  October  25,  salep  was  isolated,  which  yielded  on 
hydrolysis  0.39  grams  reducing  sugar  as  dextrose;  it  was  also  isolated 
from  the  urines  of  the  next  two  days,  but  was  not  estimated  quanti- 
tatively. 

Experiment  C.  A  dog  weighing  9.2  kg.  received  90  cc.  of  salep 
solution,  containing  1.8  grams  of  pure  mannan.  No  reduction  of 
Fehling's  solution  occurred  with  any  of  the  samples.  Tests  for  salep 
with  Fehling's  solution  gave  the  following  results: 


Examination  of  Urine. 


TIME. 

VOLUME. 

ROTATION. 

SALEP  PRESENT. 

December  2,  10  A.M  

CC. 

-0.17° 

December  2,2:30P.M  

Injection 

December  3,  10  A.M  

960 

+ 

December  4, 10  A.M  

234 

-  0.41° 

+  (0.6gm.) 

December  5,  10  A.M  

520 

-  0.27° 

+  (0.5gm.) 

Unfortunately  this  experiment  was  unavoidably  interrupted  at 
this  point.  The  salep  was  precipitated  from  50  cc.  of  the  urine  for 
December  3,  hydrolyzed,  and  sugar  determined  gravimetrically  as 
dextrose,  from  which  the  total  amount  of  salep  in  this  day's  urine  was 
calculated  as  0.67  gram.  Salep  determined  in  the  same  way  on  De- 
cember 4,  showed  an  elimination  of  0.18  gram;  hence  0.85  gram  was 
actually  recovered  in  these  two  days.  The  influence  of  the  levo- 
rotatory  carbohydrate  on  the  rotation  of  the  urine  was  marked. 


340 


Mary  Dames  Swartz, 


Experiment  D.  A  dog  weighing  6.4  kg.  received  98  cc.  of  salep 
solution  containing  1  gram  of  pure  mannan.  No  reduction  of  Feh- 
ling's  solution  was  observed  throughout  the  experiment.  The  changes 
in  rotation  due  to  the  salep  are  shown  in  the  following  table: 


Examination  of  Urine. 


TIME. 

VOLUME. 

ROTATION. 

SALEP    PRECIPITATED  BY 
FEHLING'S  SOLUTION. 

February  1  

CC. 

116 

Injection 
152 
238 
154 
137 

-  0.41° 

-  0.41° 

-  0.13° 

-  0.13° 

-  0.20° 

+ 

The  results  in  this  experiment  are  very  puzzling.  The  normal  rota- 
tion was  high  (  —  0.41°)  for  several  weeks  before  this  experiment 
but  fairly  constant,  averaging  —0.44°.  If  salep  were  excreted  as 
mannan,  the  levo-rotation  should  have  increased,  yet  it  was  decid- 
edly low  on  a  day  when  salep  was  shown  to  be  present,  and  also  on  a 
day  when  none  could  be  detected.  The  absence  of  any  positive  tests 
for  sugar,  excluded  the  idea  that  the  salep  was  being  excreted  in  this 
form,  but  finally  a  sample  of  February  4,  was  tested  with  yeast,  and 
marked  fermentation  observed.  Unfortunately,  this  was  after  all  the 
other  samples  had  been  discarded,  hence  no  further  tests  could  be 
made. 

Experiment  E.  A  dog  weighting  9.8  kg.  received  intraperitoneally 
97.5  cc.  of  salep  solution  containing  1.3  grams  pure  mannan.  No  re- 
duction of  Fehling 's  solution  was  observed.  The  changes  in  rotation 
are  shown  in  the  first  table  on  the  next  page. 

Salep  was  isolated  and  identified  as  carbohydrate,  in  the  urines  of 
May  19,  20,  and  21,  although  the  amount  in  the  last  two  days  was  ap- 
parently too  small  to  be  detected  by  any  change  in  the  rotation. 

INJECTIONS  OF  SINISTRIN. 

i.  Subcutaneous. 

A  dog  weighing  6.5  kg.  received  49  cc.  of  sinistrin  solution,  contain- 
ing 3.3  grams  pure  substance.    This  solution  showed  a  rotation  of 


Nutr  ition  Investigations . 


341 


Examination  of  Urine. 


TIME. 

VOLUME. 

ROTATION. 

ESTIMATION   OF  AMOUNT 
OF  SALEP  EXCRETED. 

CC. 

Grams. 

May  17  10  A  M 

—  0. 14° 

May  18,  10A.M  

-  0.14° 

May  18,  3  P.M  

Injection 

May  19,  9  A.M  

165 

-  0.34° 

0.4 

May  20,  9  A.M  

250 

-  0 . 14° 

Salep  present — pre- 

cipitated by  Fen- 

ling's  Solution. 

May  21, 11  A.M  

405 

-  0.14° 

Salep  present. 

May  22,  9  A.M  

200 

-  0.14° 

No  Salep  present. 

—  3.88°  in  a  200  mm.  tube.  The  urine  contained  no  reducing 
substance  at  any  time.  The  changes  in  rotation,  due  to  sinistrin  in- 
jection, are  shown  in  the  following  table: 


Examination  of  Urine. 


TIME. 

VOLUME. 

ROTATION. 

ESTIMATION  OF  AMOUNT 
OF  SINISTRIN  EXCRETED.* 

cc. 

Grams. 

January  15, 12  M  

-  0.41° 

January  15,2:30  P.M  

Injection 

260 

-  0.97° 

2.5 

January  17  and  18  

165 

-  0.41° 

60 

-  0.41° 

108 

-  0.47° 

*  Calculating  for  sinistrin  [a]  d  =  — 

29.1°. 

2.  Intraperitoneal. 

Experiment  A.  A  dog  weighing  6.5  kg.  received  110  cc.  of  sinis- 
trin solution,  containing  2  grams  pure  substance.  This  solution 
showed  a  rotation  of  —1.18°  in  a  200  mm.  tube.  No  reducing  sub- 
stance was  found  in  the  urines  examined.  The  changes  in  rotation, 
due  to  sinistrin  injection,  are  shown  in  the  following  table: 


342 


Mary  Davies  Swartz, 
Examination  of  Urine. 


January  11,  10:30  A.M  

January  11,  3  P.M  

January  12,9:30  A.M  

January  13,  9:30  A.M  

January  14,9:30  A.M  

*  Calculating  for  sinistrin  [a]  d  =  —  29.1°. 


Injection 
88 
127 
116 


-0.48° 

-  2.04° 

-  0.48° 

-  0.48° 


ESTIMATION  OF  AMOUNT 
OF  SINISTRIN  EXCRETED.  * 


Experiment  B.  A  dog  weighing  4.6  kg.  received  108  cc.  of  sinis- 
trin solution,  containing  2.3  grams  pure  substance.  The  rotation  of 
this  solution  was  —1.38°  in  a  200  mm.  tube.  No  reducing  sub- 
stance was  detected  in  the  urine  at  any  time.  The  changes  in  rota- 
tion are  shown  in  the  following  table : 


Examination  of  Urine. 


TIME. 

VOLUME. 

ROTATION. 

ESTIMATION   OF  AMOUNT 
OF  SINISTRIN  EXCRETED.* 

CC. 

Crams. 

January  26  

-  0.14° 

January  27,9:30  A.M  

Injection 

January  27,  5:P.M  

148 

-  1.38° 

2.1 

January  28,  9  :AM  

95 

-  0.41° 

0.4 

January  29,  9: A.M  

155 

-  0.14° 

*  Calculating  for  sinistrin  la]  D  =  —  29.1°. 


In  all  these  experiments,  the  sinistrin  was  isolated  and  identified  as 
a  levo-rotatory  carbohydrate,  yielding  reducing  sugar  on  hydrolysis. 
It  was  apparently  excreted  quantitatively  in  every  case. 


FEEDING  EXPERIMENTS. 


Methods  and  Technique. 

Feeding  experiments  were  conducted  with  dogs  and  human  sub- 
jects, under  conditions  as  nearly  normal  as  possible.  The  dogs  were 
kept  in  metal  cages,  arranged  for  the  separate  collection  of  urine  and 
faeces.  They  were  fed  once  a  day,  on  a  uniform  weight  diet,  consist- 
ing of  chopped  lean  meat,  lard,  and  cracker  meal,  in  suitable  portions 


Nutrition  Investigations. 


343 


and  amounts  to  maintain  a  constant  body  weight.  The  carbohydrate 
under  investigation  was  dissolved  or  suspended  in  water,  and  mixed 
with  this  basal  ration.  In  the  earlier  experiments  the  periods  were 
divided  as  follows :  Fore  =  3  days  on  the  basal  ration ;  mid  =  3  days 
in  which  some  preparation  was  added,  the  amount  being  the  same 
each  day ;  after  =  3  days  like  the  fore  period.  Separation  of  the  pe- 
riods in  the  faeces  was  accomplished  by  marking  with  soot  or  carmine 
capsules.  In  all  later  experiments,  two  days  constituted  the  fore  pe- 
riod, and  a  day  on  the  normal  diet  was  included  at  the  beginning  and 
end  of  the  mid  period,  making  thus  four  days,  to  insure  against  any 
of  the  material  under  investigation  being  carried  into  the  faeces  of 
the  after  period. 

In  several  cases,  the  presence  or  absence  of  galactans  or  mannans 
in  the  faeces  has  been  verified  by  testing  the  hydrolyzed  material  for 
mucic  acid  or  mannose-hydrazone. 

For  analysis,  the  faeces,  collected  and  weighed,  were  rubbed  to  a 
thin  mud  with  alcohol,  dried  to  constant  weight  on  a  water  bath, 
weighed  air  dry,  and  ground  finely  in  a  coffee  mill.  The  portions 
constituting  each  period  were  thoroughly  mixed,  and  from  2  to  5 
grams  taken  for  hydrolysis,  according  to  the  yield  of  carbohydrate 
anticipated.  The  samples  were  boiled  on  a  reflex  condenser  with 
100  cc.  of  2  per  cent  hydrochloric  acid,  for  two  hours;  or  longer  if 
thought  to  contain  a  carbohydrate  which  previous  analysis1  had 
shown  to  require  more  time  for  complete  hydrolysis. 

The  products  of  hydrolysis,  cooled  and  neutralized,  were  made  up 
to  250  cc.  and  sugar  determined  as  dextrose  by  Allihn's  gravimetric 
method.  It  was  found  that  the  copper  reduction  was  often  very  in- 
complete, and  that  much  more  satisfactory  results  came  from  clari- 
fying the  solutions  with  charcoal  after  making  up  to  volume.  Not 
only  were  duplicate  analyses  in  closer  agreement,  but  in  some  cases 
the  yield  of  cupric  oxide  was  two  or  three  times  greater  than  before 
this  treatment.  Owing  to  the  complexity  and  diversity  of  the  prod- 
ucts of  hydrolysis,  results  are  at  best  only  approximate. 

In  experiments  with  dulse,  the  pentosans  were  determined  by  the 
phloroglucin  method.2 

The  human  subjects  were  healthy,  active  young  women.  Their 
diet  was  not  weighed,  but  was  kept  as  uniform  as  possible.    All  cel- 


1  Cf .  table,  p.  317. 

2Cf.  Official  and  Provisional  Methods  of  Analysis,  Bulletin  No.  107  (1907), 
Bureau  of  Chemistry,  United  States  Department  of  Agriculture. 


344 


Mary  Dames  Swartz, 


lulose-containing  foods,  such  as  nuts,  fruits,  green  vegetables,  peas 
and  beans,  coarse  bread  and  cereals,  were  carefully  avoided;  so  that 
the  carbohydrates  were  limited  almost  entirely  to  bread  and  crackers 
made  from  fine  white  flour,  a  small  quantity  of  potato,  and  sugar. 
To  this  diet  the  gelatinizing  carbohydrates  were  added  in  the  form  of 
blanc  mange  or  jelly;  dulse  was  dissolved  in  some  beverage,  and  the 
insoluble  preparations  boiled  half  an  hour  in  a  little  water  and  eaten 
as  a  vegetable,  seasoned  with  salt,  butter,  and  vinegar.  The  blanc 
manges  or  jellies  made  from  the  Hawaiian  seaweed  preparations  were 
equally  attractive  in  texture  and  flavor  with  those  made  from  Irish 
moss. 

Periods  were  marked,  and  the  analyses  of  faeces  conducted  in  the 
manner  already  described  for  the  experiments  with  dogs. 

The  Digestibility  of  Pentosans. 

Four  preparations  were  fed,  Dulse,1  Limu  Eleele,2  Limu  Lipoa,2 
and  Limu  Pahapaha,3  without  production  of  unpleasant  symptoms  in 
any  case.  The  results  of  all  trials  are  shown  in  the  tables  on  the 
following  pages. 


1  Cf.  p.  303. 

2  Cf.  p.  307. 

3  Cf.  p-  308. 


3  §2 
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Grams. 

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3.8 
2.1 

Composition 

Per  cent. 

4.8 

8.1 
8.2 

WEIGHT 
AIR  DRY. 

Grams. 

22.5 

46.4 
26.8 

WEIGHT 
MOIST. 

Grams. 

65.7 

95.4 
193.4 

diet. 

Cellulose-free 

Same  +  30  gms.  Limu  Paha- 

paha,  boiled  |  hr. 
Same  as  Fore  Period 

PERIOD. 

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348 


Mary  Dames  Swartz, 


The  coefficients  of  digestibility  of  the  pentosan  preparations,  as 
determined  in  the  usual  way  from  the  preceding  experiments,  are  set 
forth  in  the  following  table: 


SERIES  A. 

PENTOSAN. 

COEFFICIENT  OF 

DIGESTIBILITY. 

EXPERIMENT  NO. 

For  the  Dog. 

For  Man. 

1 

Dulse 

80 

2 

Dulse 

66 

3 

Dulse 

100 

4 

Dulse 

100 

5 

Limu  Eleele 

50 

6 

Limu  Eleele 

20 

7 

Limu  Eleele 

69 

8 

Limu  Pahapaha 

34 

9 

Limu  Lipoa 

16 

It  is  evident  from  these  figures,  that  pentosans  in  soluble  form  dis- 
appear from  the  alimentary  tract  of  dogs  to  a  very  considerable  extent 
(average  73  percent),  and  that  small  quantities,  ingested  by  man,  do 
not  reappear  in  the  faeces.  The  insoluble  limu  preparations  appear 
much  more  indigestible,  an  average  of  28  per  cent  being  digested  by 
dogs,  and  51  per  cent  by  man. 

It  must  be  borne  in  mind,  in  interpreting  the  results  of  these  metabo- 
lism experiments,  that  they  are  at  best  only  approximate.  The  dif- 
ficulty of  strict  separation  of  the  faeces,  the  fact  that  the  human  sub- 
jects were  not  kept  on  a  uniform  weighed  diet,  and  the  errors  unavoid- 
ably introduced  by  determining  many  different  kinds  of  sugar  as  dex- 
trose, make  all  of  the  figures  given  as  "coefficients  of  digestibility," 
in  this  and  succeeding  sections,  comparative  rather  than  absolute. 

The  Digestibility  of  Galactans. 

In  these  experiments,  preparations  of  the  water  extracts  of  Irish 
moss,  Limu  Manauea,  Limu  Huna  and  Limu  Akiaki  have  been  fed, 
without  any  disagreeable  symptoms.  The  results  are  given  in  the 
tables  which  follow: 


7 


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352 


Mary  Dames  Swartz, 


The  coefficients  of  digestibility  of  the  galactan  preparations  are  given 
in  the  following  table: 


SERIES  B. 
EXPERIMENT  NO. 

GALACTAN. 

COEFFICIENT  OI 

For  the  Dog. 

DIGESTIBILITY. 

For  Man. 

Per  cent. 

Per  cent. 

1 

Irish  Moss 

46 

2 

XI  loll  itlUjo 

3 

Irish  Mioss 

H 

4 

Alloil 

0 

5 

Limu  Manauea 

55 

6 

Limu  Manauea 

12 

7 

Limu  Manauea 

30 

8 

Limu  Manauea 

30  (av.) 

9 

Limu  Huna 

30  (20  gms. 

fed) 

10 

Limu  Huna 

83  (7  gms. 

fed) 

11 

Limu  Huna 

10 

12 

Limu  Akiaki 

60 

Although  these  preparations  were  administered  in  small  quanti- 
ties, under  the  most  favorable  conditions  for  digestion  in,  the  only 
instance  where  the  utilization  in  any  degree  approaches  that  of  starch 
(Limu  Huna) ,  the  quantity  fed  (7  grams)  was  so  small  that  this  exper- 
iment can  hardly  be  taken  as  a  criterion  of  digestibility.  Exclusive 
of  this  experiment,  the  average  of  five  trials  with  dogs  is  32  per  cent, 
while  that  of  six  trials  with  human  subjects  is  23  per  cent.  In  both 
cases,  the  averages  are  lower  than  that  of  Lohrisch  (194)  for  "  soluble 
agar,"  50  per  cent. 

Where  the  quantity  of  galactan  fed  was  10  or  more  grams,  the  in- 
fluence on  the  character  of  the  faeces  was  usually  noticeable.  The 
increase  in  bulk,  after  ingestion  of  45  grams  of  Irish  moss,  is  well  illus- 
trated in  a  photograph  of  the  dried  and  ground  faeces  of  the  dogs 
used  in  experiments  1  and  2:1 

A  represents  the  fore-period  (3  days),  B  the  mid-period,  during  which 
15  grams  of  moss  were  ingested  daily  (3  days),  and  C  the  after-period 
(3  days).  The  separation  of  the  faeces  at  the  beginning  of  experi- 
ment 1  (on  the  right)  was  not  very  satisfactory.  The  dog  had  pre- 
viously been  fed  bone-ash,  and  the  marked  faeces  were  undoubtedly 
contaminated  with  this,  so  that  they  appear  unusually  bulky.  Exper- 
iment 2  is  typical  of  the  results  obtained  in  most  of  the  experiments 


1  Cf.  p.  343. 


Nutrition  Investigations. 


353 


with  human  subjects.  In  these,  the  undigested  hemicelluloses  gave 
frequently  a  peculiar,  wax-like  consistency,  especially  noticeable  with 
imu  Huna  in  the  experiment  recorded,1  and  in  another  not  reported, 
ecause  the  faeces  for  part  of  the  time  were  lost.  In  the  experiment 
with  Limu  Akiaki  (No.  12), 1  the  galactan  was  excreted  after  the  first 
day's  feeding,  in  a  tough  mass  almost  impossible  to  break  up  with  a 


EXP.  2 


The  Influence  of  Irish  Moss  upon  the  Mass  of  the  Faeces. 

A.  Fore  Period:    3  Days  on  a  Cellulose-free  Diet. 

B.  Mid  Period:    3  Days  on  a  Cellulose-free  Diet  to  Which  15  grams  of 
Irish  Moss  were  Added  Daily. 

C.  After  Period:  3  Days  of  a  Cellulose-free  Diet. 

spatula.  That  of  the  second  day  was  not  excreted  till  the  third 
day  after  feeding,  the  subject  being  inclined  to  constipation.  It  seems 
likely  that  the  high  coefficient  of  digestibility  is  due  to  this  fact,  or 
else  to  the  method  of  determination,  which  is  not  altogether  satisfac- 
tory, in  view  of  the  complexity  of  the  products  of  hydrolysis,  the  dan- 
ger of  decomposing  a  part  of  the  sugar  from  the  easily  inverted 
polysaccharides  by  the  long  boiling  necessary  for  the  more  resistant, 
and  the  great  difference  in  reducing  power  of  the  sugars  so  produced. 

The  Digestibility  of  Mannan. 

In  four  experiments,  the  commercial  salep  powder  (containing  19 
per  cent  mannan  and  26  per  cent  starch)  was  administered;  in  the 
others,  pure  mannan  prepared  from  the  Orchis  tubers.  The  results 
of  seven  trials  are  tabulated  on  the  following  pages. 


1  Cf.  p.  345. 


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356 


Mary  Davies  Swartz, 


The  coefficients  of  digestibility  of  the  salep  preparations  are  shown 
in  the  following  table : 


SERIES  C. 
EXPERIMENT  NO. 


COEFFICIENT  OF  DIGESTIBILITY. 


For  the  Dog. 


Per  cent 

Per  cent 

1 

Salep  Powder 

70 

2 

Salep  Powder 

100 

3 

Salep  Powder 

100 

4 

Salep  Powder 

94 

5 

Salep  Mannan 

10 

6 

100 

7 

100 

For  Man. 


Thus  we  see  that  in  every  case,  except  that  in  which  a  dog  received, 
in  one  day,  10  grams  of  pure  mannan,  the  greater  portion  of  the  salep 
fed  was  digested,  the  coefficient  of  salep  powder  for  dogs  averaging 
85  per  cent,  and  for  man,  97  per  cent;  while  that  of  pure  mannan  for 
man  is  100  per  cent,  in  spite  of  the  fact  that  it  is  not  attacked  by  diges- 
tive enzymes! 

The  contrast  between  the  volume  of  faeces  produced  when  a  galac- 
tan  such  as  Irish  moss  was  fed,  and  that  when  a  more  digestible  hemi- 
cellulose  was  given,  is  shown  in  the  photograph  of  the  faeces  from 
experiments  Nos.  1  and  2  of  Series  C,1  on  the  next  page,  in  which  A 
represents  the  fore-period,  B  the  mid-period,  and  C  the  after-period, 
each  period  being  three  days  in  duration.  The  group  on  the  right 
represents  experiment  No.  1,  in  which  70  per  cent  of  the  hemicellulose 
and  starch  of  the  salep  powder  was  digested,  and  that  on  the  left, 
experiment  No.  2  in  which  apparently  all  of  these  were  digested. 


DISCUSSION  AND  SUMMARY. 


A  glance  at  the  table  on  page  327  clearly  shows  that  none  of  the 
hemicelluloses  under  consideration  are  readily  attacked  by  the  ordi- 
nary animal  or  vegetable  enzymes.  The  results  are  for  the  most  part 
entirely  negative.  Even  where  there  has  been  hydrolysis  with  0.2 
per  cent  hydrochloric  acid,  the  amount  of  sugar  produced  in  24  hours 
was  relatively  small.  The  hydrolyzing  action  of  the  gastric  juice 
is  probably  largely  due  to  the  presence  of  acid,  although  no  compari- 
son of  the  relative  amounts  of  sugar  produced  by  gastric  juice  or  by 


lCf.  p.  348. 


Nutrition  Investigations.  357 

0.2  per  cent  acid  alone  has  been  made.  It  is  noticeable  that  even  the 
very  soluble  hemicellulose,  sinistrin,  which  is  so  speedily  hydrolyzed 
by  acid  (in  §  hour  at  37°  C.  with  0.2%  hydrochloric  acid)  is  not  attacked 
by  ordinary  diastatic  enzymes  within  24  hours. 

The  parenteral  introduction  of  these  carbohydrates  has  resulted  in 
their  speedy  and  apparently  complete  elimination  through  the  kid- 
neys without  any  change  in  character.  The  carbohydrates  prepared 
from  Dulse,  Irish  Moss,  Salep  and  Sinistrin  have  all  been  isolated  and 
identified  in  the  urine,  after  subcutaneous  and  intraperitoneal  injec- 
tions.   These  results  are  not  surprising,  in  view  of  the  commonly  ac- 


The  Influence  of  Salep  upon  the  Mass  of  the  Faeces. 

A.  Fore  Period:    3  Days  on  a  Cellulose-free  Diet. 

B.  Mid  Period:    3  Days  on  a  Cellulose-free  Diet  to  Which  15  grams  of 

Salep  Powder  were  Added  Daily. 

C.  After  Period:  3  Days  on  a  Cellulose-free  Diet. 

cepted  fact  that  carbohydrates  must  be  converted  into  monosacchar- 
ides before  they  can  enter  into  the  processes  of  intermediary  meta- 
bolism. 

Experimental  evidence  in  support  of  this  fact  is  given  by  such 
investigators  as  F.  Voit1  and  Blumenthal,2  who  found  that  even  di- 
saccharides,  as  lactose  and  saccharose,  were  eliminated  almost  quanti- 


^iinchener  medicinische  Wochenschrift,  1896,  p.  717;  Deutsches  Archiv  fur 
klinische  Medicin,  v.  58,  p.521  (1897). 

2Beitage  zur  chemischen  Physiologie,  v.  6,  p.  329  (1905). 


358 


Mary  Dames  Swartz, 


tatively  after  subcutaneous  injection  in  man  and  the  rabbit;  or  as 
Mendel  and  Mitchell,1  who  have  shown  that  polysaccharides  like 
dextrin,  soluble  starch,  glycogen,  inulin,  and  isolichenin  are  recovered 
to  a  considerable  extent  in  the  urine,  whether  introduced  subcuta- 
neously,  intraperitoneally,  or  intravenously. 

In  the  present  experiments,  the  dulse  pentosan  was  the  most  slowly 
eliminated,  being  found  in  the  urine  four  or  five  days  after  injection; 
Irish  moss  and  salep  were  not  detected  after  the  third  day;  while  si- 
nistrin  seemed  to  be  all  excreted  within  the  first  24  hours. 

The  average  coefficients  of  digestibility  for  the  ten  preparations 
which  have  formed  the  basis  of  the  feeding  experiments,  are  summar- 
ized in  the  following  table : 


Coefficients  of  Digestibility  of  Hemicelluloses. 


HEMICELLULOSE. 


Class. 

Source. 

Dog. 

Man. 

Per  cent. 

Per  cent. 

Pentosan  

Dulse 

73  (2  exp.) 

100  (2  exp.) 

Pentosan  

Limu  Eleele 

35  (2  exp.) 

9  (2  exp.) 

Pentosan  

Limu  Pahapaha 

34  (1  exp.) 

Pentosan  

Limu  Lipoa 

16  (1  exp.) 

Galactan  

Irish  Moss 

33  (2  exp.) 

6  (2  exp.) 

Galactan  

Limu  Manauea 

33  (2  exp.) 

30  (3  exp.) 

Galactan .  

Limu  Huna 

56  (2  exp.) 

10  (1  exp.) 

Galactan  

Limu  Akiaki 

60  (1  exp.)* 

Mannan  

Salep  Powder 

85  (2  exp.) 

97  (2  exp.) 

Salep  Mannan 

10  (1  exp.) 

100  (2  exp.) 

SUBJECT  OF  EXPERIMENT. 


Subject  with  chronic  constipation. 


That  the  low  coefficients  enumerated  above  are  not  due  to  inabil- 
ity of  the  various  subjects  to  utilize  carbohydrates,  is  shown  by  the 
following  figures. 

The  coefficient  of  digestibility  for  cracker  meal  in  the  experiments 
on  dogs,  determined  by  taking  the  average  of  all  the  fore-periods  of 
the  feeding  trials,  in  which  five  different  dogs  were  used,  was  99.0 
per  cent.  This  is  much  higher  than  London  and  Polowzowa's2  coef- 
ficient for  carbohydrate  digestibility  in  dogs  on  a  bread  diet,  96  per 
cent. 


1  American  Journal  of  Physiology,  v.  14,  p.  239  (1905). 
2Zeitschrift  fur  physiologische  Chemie,  56,  513  (1908). 


Nutrition  Investigations. 


359 


For  the  four  women  who  were  subjects  of  feeding  experiments,  the 
average  daily  amount  of  carbohydrate  excreted  in  the  faeces,  on  a 
cellulose-free  diet,  estimated  as  dextrose,  by  averaging  the  fore-pe- 
riods of  all  trials,  was  0.8  gram.  The  utilization  of  carbohydrates  was 
therefore  unusually  good,  since  Atwater  and  Bryant's1  coefficient  of 
digestibility  for  such  a  diet  is  98  per  cent,  and  undoubtedly  every  one 
of  these  individuals  consumed  over  50  grams  of  carbohydrate  per  day. 
With  the  exception  of  the  subject  of  a  single  experiment  who  had 
chronic  constipation,  these  were  all  normal,  healthy  individuals,  free 
from  disturbances  of  the  alimentary  tract. 

The  three  seaweeds  fed  in  toto,  Limu  Eleele,  Limu  Pahapaha,  and 
Limu  Lipoa,  show  an  average  digestibility  of  51  per  cent.  This  is 
higher  than  that  obtained  in  Professor  Mendel's  laboratory  for  un- 
cooked Cetraria  islandica2  (average  of  three  experiments,  15  per  cent) 
and  much  lower  than  that  reported  by  Oshima  for  dried  marine  algae3 
(average  77  per  cent). 

In  man,  with  the  exception  of  dulse  and  salep,  which  almost  entirely 
disappeared  in  the  alimentary  tract,  the  average  digestibility  of  all 
preparations  is  only  34  per  cent,  a  figure  in  contrast  to  those  of  Loh- 
risch  (194),  who  finds  cellulose  and  hemicellulose  50  per  cent  digestible. 
In  dogs,  the  average  of  all  preparations  is  42  per  cent. 

Considering  that  the  pentosan  of  dulse  was  in  a  form  most  favorable 
for  digestion,  the  results  with  this  hemicellulose  are  in  harmony 
with  those  of  Konig  and  Reinhardt  (120)  who  reported  75  per  cent  of 
the  pentosans  as  disappearing  from  the  alimentary  tract  in  man; 
and  with  the  averages  obtained  by  the  various  investigators  on  ani- 
mals, which  show  these  carbohydrates  40-70  per  cent  diges- 
tible in  herbivora.4  It  would  be  desirable  to  repeat  the  experiments 
with  larger  quantities,  although  the  process  of  preparing  the  material 
is  rather  laborious.  It  must  be  borne  in  mind,  that  the  dulse  pento- 
san is  not  attacked  by  ordinary  diastatic  enzymes,  but  can  be  decom- 
posed by  soil  and  faecal  bacteria;  and  although  this  decomposition 
did  not  occur  readily  in  pure  solutions  of  the  carbohydrate,  or  even 
in  a  putrefying  mixture,  it  still  remains  to  be  demonstrated  whether 
the  complete  disappearance  from  the  alimentary  tract  is  not  largely 
due  to  the  more  favorable  conditions  for  bacterial  activity  within  the 


1  Report  Storr's  Agricultural  Experiment  Station,  1899,  p.  86. 

2  Cf .  pp.  297-298. 

3  Cf.  p.  299. 

4  Cf.  pp.  274-275. 


360 


Mary  Davies  Swartz, 


organism.  While  we  have,  in  the  case  of  herbivora,  some  convincing 
evidence  that  the  pentosans  are  a  true  source  of  energy,1  we  have  as 
yet  no  real  grounds  for  this  assumption  in  the  case  of  man. 

The  insoluble  pentosans  of  the  Hawaiian  algae  are  manifestly  less 
digestible  than  the  soluble  forms.  The  coefficient  of  digestibility  is 
approximately  the  same  as  Slowtzoff's  (154)  average  for  pure  xylan 
in  rabbits,  55  per  cent.  While  it  would  be  perhaps  desirable  to  de- 
termine the  pentosans  directly  by  the  furfurol-phloroglucin  method, 
rather  than  by  estimation  of  sugar  after  acid  hydrolysis,  a  trial  with 
dulse  by  both  methods  gave  practically  identical  results:  hence,  con- 
sidering that  the  hemicelluloses  of  these  algae  are  chiefly  pentosans, 
it  seems  safe  to  assume  that  the  results  reported  represent  the  amount 
of  pentosan  excreted,  within  the  limits  of  error  for  all  of  the  feeding 
experiments. 

The  galactans  were  all  soluble,  and  were  ingested  in  quantities  not 
exceeding  15  grams  per  day,  yet  the  coefficient  of  digestibility  is  lower 
than  for  any  other  hemicellulose  group  (26  per  cent) .  The  resistance 
of  Irish  moss  is  particularly  striking,  but  is  not  surprising  in  view  of 
its  utter  indifference  to  attacks  of  digestive  enzymes  or  bacteria.  Its 
influence  on  the  character  of  the  faeces  was  not  so  marked  as  that  of 
Limu  Huna,  owing  probably  to  a  greater  tendency  to  liquefy  at  body 
temperature.  The  latter  would  seem  to  be  a  very  effective  agent  in 
constipation;  a  comparison  of  its  efficiency  with  that  of  agar-agar 
would  be  extremely  interesting.  Saiki  (205)  found  the  coefficient  of 
digestibility  for  agar  (average  of  two  experiments)  17  per  cent. 

In  view  of  the  negative  results  of  digestions  in  vitro  and  of  trials 
with  bacteria,  we  can  scarcely  be  surprised  at  the  results  of  these  met- 
abolism experiments,  especially  as  we  recall  that  Lohrisch  (57)  found 
that  his  "soluble  agar,"  already  partially  hydrolyzed,  was  only  diges- 
tible to  50  per  cent  (average). 

The  mannans  stand  in  striking  contrast  to  the  galactans.  In  the 
present  studies,  99  per  cent  of  the  salep  administered  has  been  uti- 
lized, a  result  in  accordance  with  Kano  and  Iishima's  (255)  coefficient 
of  digestibility  for  the  Japanese  mannan,  Konjaku,  82  per  cent. 
Pure  mannan  fed  to  a  dog,  was  excreted  the  succeeding  day,  seem- 
ingly unaltered,  since  it  formed  a  semi-transparent  gelatinous  mass  in 
the  faeces,  from  which,  later,  a  rich  yield  of  mannose-hydrazone  was 
obtained.  The  very  different  result  with  salep  powder,  of  which  85 
per  cent  was  digested  by  dogs,  may  perhaps  be  accounted  for  by  the 


3  Cf.  Kellner,  p.  274. 


Nutrition  Investigations. 


361 


fact  that  it  contained  a  high  percentage  of  starch  (26  per  cent).  The 
amount  of  undigested  carbohydrate  excreted  in  the  faeces  is  in  close 
agreement  with  the  quantity  of  pure  mannan  ingested.  However, 
as  tests  for  mannose-hydrazone  were  negative  in  these  cases,  further 
experiments  are  necessary  before  an  authoritative  statement  can  be 
made  in  regard  to  this  question. 

It  is  manifestly  possible  for  faecal  and  soil  bacteria  to  produce 
sugar  from  mannan;1  hence  it  is  not  unlikely  that  hemicelluloses  of 
this  group  are  inverted  in  the  intestines  through  the  activity  of  micro- 
organisms, and  that  the  sugar  so  produced  is  absorbed  and  becomes  a 
true  source  of  energy  for  man,  in  spite  of  the  resistance  of  mannans  to 
the  action  of  digestion  enzymes.  Further  investigations  to  determine 
its  exact  nutritive  value  seem  highly  desirable. 

In  considering  the  proper  place  in  the  dietary  for  marine  algae, 
lichens  and  similar  substances,  we  must  not  disregard  the  possibility 
of  their  having  a  valuable  function  entirely  aside  from  the  question 
of  energy  production.  As  Oshima  (15)  points  out,  they  may  be  val- 
uable for  their  inorganic  salts.  The  non-irritating,  laxative  proper- 
ties of  many  species  make  them  desirable  adjuncts  to  the  diet  of  per- 
sons with  a  tendency  to  constipation;2  and  even  if  they  disappear,  in 
marked  degree,  from  the  alimentary  tract  during  the  process  of  diges- 
tion, they  may  perhaps  still  play  an  important  role  as  stimulants  to 
intestinal  activity,  being  in  fact  what  Prausnitz3  calls  "faeces-forming 
foods."  An  illustration  of  this  effect  is  afforded  by  the  experiments 
in  which  salep  powder  was  fed  to  dogs.4  The  periods  were  equal  in 
length,  and  in  one  case  (No.  2  in  photograph)  the  utilization  of  carbo- 
hydrates was  equally  good  for  all  three;  yet  in  the  mid-period  there 
is  a  decided  increase  in  the  bulk  and  weight5  of  the  faeces,  not  more 
than  1  gram  of  which  is  by  any  possibility  attributable  to  the  cellu- 
lose of  the  salep  powder,  and  in  the  other  experiment,  the  increased 
amount  of  faeces  cannot  be  wholly  accounted  for  by  the  amount  of 
undigested  carbohydrate  present. 

Mendel  (196)  has  already  sounded  a  warning  against  the  hasty 
assumption  that  every  carbohydrate,  by  virtue  of  its  ultimate  chem- 
ical composition,  stands  in  the  category  of  true  nutrients  for  the  human 
organism.    The  results  of  the  present  investigations  emphasize  the 

1  Cf .  Sawamura  (267) . 
2Cf.  p.  283. 

3Zeitschrift  fur  Biologie,  v.  35,  p.  335  U897). 
4Cf.  pp.  348-349. 

6  Cf.  Table,  p.  348,  Series  C,  Experiments  Nos.  1  and  2. 


362 


Mary  Dames  Swartz, 


necessity  of  drawing  our  final  conclusions  only  from  exact  metabolism 
experiments.  The  soluble  hemicelluloses  show  great  diversity  of 
behavior  in  the  alimentary  tract,  although  equally  resistant  to  diges- 
tive enzymes  in  vitro;  some  disappear  entirely,  others  reappear  in  the 
faeces  in  varying  degree,  up  to  100  per  cent.  It  is  evident  that  the 
latter  do  not  constitute  a  source  of  energy  for  the  organism:  how  far 
the  former  actually  do  so,  remains  to  be  demonstrated. 


IV.  CONCLUSIONS. 


1.  The  hemicelluloses  of  the  ten  species  of  marine  algae  included 
in  these  investigations  are  chiefly  pentosans  and  galactans.  The  pen- 
tosans are  largely  insoluble  in  water,  but  a  soluble  form  in  consider- 
able quantity  has  been  isolated  from  Rhodymenia  palmata.  The  ga- 
lactans are  soluble  in  hot  water,  and  are  characterized  by  their  gela- 
tinous nature.  Small  quantities  of  soluble  pentosans  have  been  found 
associated  with  them  in  every  case. 

2.  In  order  of  resistance  to  the  action  of  bacteria,  the  hemicellu- 
lose  groups  studied  stand  as  follows,  —  galactans,  pentosans,  levu- 
lans,  mannans,  the  galactan  of  Chondrus  crispus  being  entirely  unaf- 
fected by  common  micro-organisms. 

3.  Aerobic  and  anaerobic  cultures  of  soil  and  faecal  bacteria,  and 
cultures  of  B.  anthracis  symptomatici  and  B.  maligni  oedematis,  caused 
inversion  of  salep  mannan,  with  actual  production  of  reducing  sugar. 
The  latter  cultures  also  hydrolyzed  the  pentosan  of  Rhodymenia  pal- 
mata, and  the  levulan,  sinistrin.  In  a  mixture  of  aerobes,  salep  ap- 
peared to  be  partially  hydrolyzed,  forming  an  insoluble  transition 
product. 

4.  Digestion  experiments  in  vitro,  continued  for  24  hours  at  body 
temperature  under  antiseptic  conditions,  have  been  almost  entirely 
negative  in  result.  The  only  exceptions  are  the  hydrolysis  of  the  pento- 
san of  dulse,  the  galactan  of  limu  kohu,  and  the  levulan,  sinistrin, 
by  "Taka"  diastase;  and  of  sinistrin,  and  the  galactans  of  limu  kohu, 
limu  akiaki,  and  slippery  elm  bark,  by  artificial  gastric  juice  or  0.2 
per  cent  hydrochloric  acid,  the  action  of  the  gastric  juice  being  in  all 
probability  due  to  its  acidity. 

5.  After  parenteral  injection,  whether  subcutaneous  or  intra- 
peritoneal, the  hemicelluloses  are  excreted  through  the  kidneys,  and 
can  be  recovered  unaltered  in  the  urine.  The  pentosan  of  dulse  is 
completely  eliminated  in  four  to  five  days,  and  the  carbohydrates  of 
Irish  moss,  salep  and  sinistrin,  in  one  to  three  days. 

6.  Feeding  experiments  show  that  those  hemicelluloses  most 
readily  attacked  by  bacteria  disappear  most  completely  from  the 
alimentary  tract.  The  average  coefficient  of  digestibility  for  man  is, 
in  the  case  of  the  pentosan  of  dulse  and  the  mannan  of  salep,  99  per 

363 


364 


Mary  Davies  Swartz, 


cent  notwithstanding  their  apparent  resistance  to  amylolytic  enzymes 
and  the  hydrolyzing  influence  of  the  gastric  juice;  their  disappear- 
ance seems  therefore  directly  attributable  to  bacterial  activity,  and 
the  possibility  of  sugar  formation  by  this  agency  having  been  demon- 
strated, it  remains  to  be  shown  by  means  of  respiration  experiments 
to  what  extent  materials  so  hydrolyzed  can  serve  as  true  nutrients  for 
the  organism.  Dogs  can  also  utilize  the  dulse  pentosan  to  a  consider- 
able degree,  but  their  power  to  digest  mannan  is  still  an  open  question. 

In  striking  contrast  to  the  above  hemicelluloses  stand  the  galac- 
tans,  with  their  high  degree  of  resistance  to  bacterial  decomposition; 
they  show  in  man,  an  average  digestibility  of  approximately  25  per 
cent,  in  dogs  of  45  per  cent.  It  is  manifestly  impossible  to  treat  of 
the  digestibility  of  hemicelluloses  as  a  class,  in  view  of  such  diversity 
in  the  groups.  Not  only  must  each  type  receive  special  considera- 
tion, but  distinction  must  be  drawn  between  soluble  and  insoluble  forms, 
as  is  illustrated  by  the  pentosans,  the  ratio  of  the  digestibility  coeffi- 
cient of  the  former  to  the  latter  being  approximately  100  to  50  in  man, 
and  75  to  25  in  dogs.  We  may,  however,  say  in  general,  that  they  disap- 
pear from  the  alimentary  tract  of  men  and  animals  to  an  extent  seem- 
ingly proportional  to  their  susceptibility  to  attacks  of  micro-organ- 
isms, and  give  little  justification  for  any  high  claims  made  for  them  as 
sources  of  energy  in  nutrition.  They  may,  however,  have  a  valuable 
function  as  adjuvants  in  the  dietary,  as  therapeutic  agents  in  consti- 
pation, or  as  sources  of  inorganic  salts. 

The  author  gratefully  acknowledges  the  helpful  suggestions  and 
criticism  freely  given  by  Professor  Lafayette  Mendel  throughout  the 
progress  of  this  work  and  the  kindly  interest  and  assistance  of  Pro- 
fessor Rettger  in  the  bacteriological  problems. 


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Nutr ition  Investigations . 


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370 


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(102)  Cremer:  Uber  das  Verhalten  einiger  Zuckerarten  im  thierischen  Organis- 
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(103)  Cremer:  Uber  die  Verwertung  der  Rhamnose  im  tierischen  Organismus, 
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(104)  Cross,  Bevan  and  Beadle:  Die  naturlichen  Oxycellulosen.  Berichte 
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(106)  Cross:  liber  die  Constitution  der  Pektinstoffe.  Ibid.,  V.  28,  p.  2609, 
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(107)  Czapek:  Zur  Biologie  der  holzbewohnenden  Pilze.  Berichte  der  deut- 
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(109)  Ebstein:  Einige  Bemerkungen  zum  Verhalten  d  i  Pentosen  im  mensch- 
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(111)  Frentzel:  Uber  Glycogenbildung  im  Thierkorper  nach  Fiitterung  mit 
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(112)  Fudakowski:  Spaltung  der  Arabinsaure  durch  Pepsin.  Berichte  der 
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(113)  Gotze  and  Pfeiffer:  Beitrage  zur  Frage  iiber  die  Bildung  resp.  das 
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(114)  Harrison:  A  Bacterial  Disease  of  Cauliflower  (Brassica  and  Oleraceae), 
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(115)  Hofmeister:  Die  quantitative  Trennung  von  Hemicellulose,  Cellulose, 
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(116)  Herzfeld:  Die  Pektinsubstanzen.  Chemisches  Centralblatt,  V.  II, 
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Nutrition  Investigations . 


371 


(117)  V.  Jacksch:  Uber  Alimentare  Pentosurie.  Zeitschrift  fur  Heilkunde- 
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(118)  Kellner  and  Kohler:  Untersuchungen  iiber  den  Stoff- und  Energie- 
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Landwirtschaftliche  Versuchs-Stationen,  V.  53,  p.  457,  (1900). 

(119)  Konig:  Die  Zellmembran  und  ihre  Bestandteile  in  chemischer  und 
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p.  55,  (1907). 

(120)  Konig  and  Reinhardt:  Ausnutzung  der  Pentosane  beim  Menschen. 
Zeitschrift  fur  Untersuchung  der  Nahrungs-  und  Genussmittel,  V.  5,  p.  110,  (1902) ; 
V.  7,  p.  729,  (1904). 

(121)  Krober:  Untersuchungen  iiber  die  Pentosanbestimmungen  mittelst 
der  Salzsaure-Phloroglucin  Methode,  nebst  einiger  Anwendungen.  Journal  fur 
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(122)  Lindemann  and  May:  Die  Verwerthung  der  Rhamnose  vom  normalen 
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(123)  Lindsey:  The  Pentosans.  Fifteenth  Massachusetts  State  Report,  p. 
69,  (1903). 

(124)  Lindsey  and  Holland:  Twelfth  Massachusetts  State  Report,  p.  175, 
(1900).    (Cited  by  Lindsey.) 

(125)  Lintner  and  Dull:  Uber  die  chemische  Natur  des  Gerstengummi. 
Chemiker  Zeitung,  V.  15,  Repertorium  p.  266,  (1891). 

(126)  McCollum  and  Brannon:  The  Disappearance  of  Pentosans  from  the 
Digestive  Tract  of  the  Cow.  Journal  of  the  American  Chemical  Society,  V.  31, 
p.  1252,  (1909). 

(127)  Neuberg:  Die  Physiologie  der  Pentosen  und  der  Glukuronsauren. 
Ergebnisse  der  Physiologie,  V.  3,  p.  421,  (1904). 

(128)  Neuberg  and  Wohlgemuth:  Uber  das  Verhalten  stereo-isomer  Sub- 
stanzen  im  Tierkorper.    Zeitschrift  fur  physiologische  Chemie,  V.  35,  p.  41,  (1902). 

(129)  Oshima  and  Tollens:  Uber  das  Nori  aus  Japan.  Berichte  der 
deutschen  chemischen  Gesellschaft,  V.  34,  p.  1422,  (1901). 

(130)  Pacault:  Sur  deux  proprietes  diastatiques  de  la  salive  de  l'escargot. 
Comptes  Rendus  de  la  Societe  de  Biologie,  V.  59,  p.  29,  (1905). 

(131)  Ravenna  and  Cereser:  Origin  and  Physiological  Function  of  Pen- 
tosans in  Plants,  Journal  of  the  London  Chemical  Society,  V.  96,  p.  1946,  (1909). 
(From  Atti  R.  Accad.  Lincei,  1909  [V]  18,  ii,  p.  177.) 

(132)  Reichardt:  Pararabin,  ein  neues  Kohlehydrat.  Berichte  der  deut- 
schen chemischen  Gesellschaft,  V.  8,  p.  807,  (1875). 

(133)  Reinhardt:  Die  Bestimmung  der  Cellulose  und  ihr  Verhalten,  sowie 
das  der  Pentosane  im  Darmcanal  des  Menschen.    Dissertation,  Munchen,  (1903) . 

(134)  Rohmann:  Einige  Beobachtungen  iiber  die  Verdauung  der  Kohlehydrate 
bei  Aplysien.    Centralblatt  fur  Physiologie,  V.  13,  p.  455,  (1899). 

(135)  Rubner:  tlber  den  Werth  der  Weizenkleie  fur  die  Ernahrung  des 
Menschen.    Zeitschrift  fur  Biologie,  V.  19,  p.  45,  (1883). 

(136)  Rudzinski:  Uber  die  Bedeutung  der  Pentosane  als  Bestandtheile  der 
Futtermittel,  insbesondere  des  Roggenstrohes.  Zeitschrift  fur  physiologische  Che- 
mie, V.  40,  p.  317,  (1904). 

(137)  Salkowski:  tlber  die  Gahrung  der  Pentosen.  Zeitschrift  ftir  physiolo- 
gische Chemie,  V.  30,  p.  478,  (1900). 


372 


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(138)  Salkowski:  Uber  das  Verhalten  der  Pentosen  im  Thierkorper.  Cen- 
tralblatt  fur  die  medicinische  Wissenschaften,  No.  11,  p.  193.  (1893). 

(139)  Salkowski:  Uber  das  Verhalten  des  Arabans  zu  Fehling'scher  Losung. 
Zeitschrift  fur  physiologische  Chemie,  V.  35,  p.  240,  (1902). 

(140)  Salkowski:  Ueber  die  Darstellung  des  Xylans.  Ibid.,  V.  34,  p.  162, 
(1901-2). 

(141)  Scheibler:  Uber  den  Pectinzucker  (Pectinose),  eine  neue  durch  Spal- 
tung  der  Metapectinsaure  entstehende  Zuckerart.  Berichte  der  deutschen  chemi- 
schen  Gesellschaft,  V.  1,  p.  108,  (1868). 

(142)  Scheibler:  Uber  das  Vorkommen  der  Arabinsaure  (Gummi)  in  den 
Zuckerriiben  und  liber  den  Arabinzucker  (Gummizucker).  Berichte  der  deut- 
schen chemischen  Gesellschaft,  V.  6,  p.  612,  (1873). 

(413)  Sherman:  The  Insoluble  Carbohydrates  of  Wheat.  Journal  of  the 
American  Chemical  Society,  V.  19,  p.  308,  (1897). 

(144)  Schmulewitsch:  Bulletin  de  l'Academie  de  St.  Petersbourg,  V.  25,  p. 
549,  (1879).    (Cited  by  Bergmann.) 

(145)  Schone  and  Tollens  :  Untersuchungen  uber  Kohlehydrate.  Die 
Landwirtschaftlichen  Versuchs-Stationen,  V.  40,  p.  377,  (1892). 

(146)  Schorstein:  Zur  Biochemie  der  Holzpilze.  Centralblatt  fiir  Bakterio- 
logie,  Abtheilung  II,  V,  9.  p.  446,  (1902). 

(147)  Schulze,  Steiger  and  Maxwell:  Zur  Chemie  der  Pflanzenzellmem- 
branen.    Zeitschrift  fiir  physiologische  Chemie,  V.  14,  p.  227,  (1892). 

-  (148)  Selliere  :  Sur  la  presence  d'une  diastase  hydrolysant  dans  le  sue  gastro- 
intestinal d'escargot.  Comptes  Rendus  de  la  Societe  de  Biologie,  V.  58,  p.  409, 
(1905). 

(149)  Selliere:  Sur  une  diastase  hydolysant  la  xylane  dans  le  tube  digestif 
de  certaines  larves  de  Coleopteres.    Ibid.,  V.  58,  p.  940,  (1905). 

(150)  Selliere:  Sur  la  presence  de  la  xylanase  chez  differents  mollusques 
gasteropodes.    Ibid.,  V.  59,  p.  120,  (1906). 

(151)  Selliere:  Sur  Pabsorption  et  la  presence  dans  le  sang  chez  l'escargot 
des  produits  de  l'hydrolyse  digestion  de  la  xylane.  Comptes  Rendus  de  la  Societe 
de  Biologie,  V.  63,  No.  36,  (1907). 

(152)  Selliere:  Sur  la  digestion  de  la  xylane  chez  quelques  mammiferes 
herbivores.    Ibid.,  V.  64,  p.  941,  (1908). 

(153)  Selliere:  Sur  la  digestion  de  la  xylane  chez  les  mammiferes.  Comptes 
Rendus  de  la  Societe  de  Biologie,  V.  66,  p.  691,  (1909). 

(154)  Slowtzoff:  Uber  das  Verhalten  des  Xylans  im  Thierkorper.  Zeit- 
schrift fiir  physiologische  Chemie,  V.  34,  p.  181,  (1901). 

(155)  Stone:  Die  Verdaulichkeit  der  Pentosane.  Berichte  der  deutschen 
chemischen  Gesellschaft,  V.  25,  p.  563,  (1892). 

(156)  Stone  and  Jones:  Verdaulichkeit  der  Pentosane.  Centralblatt  fiir 
Agriculturchemie,  V.  22,  p.  677,  (1893). 

(157)  Tollens:  "Uber  die  in  den  Pflanzenstoffen  und  besonders  den  Futter- 
mitteln  enthaltenen  Pentosane,  ihre  Bestimmungsmethoden  und  Eigenschaften. 
Journal  fiir  Landwirthschaft,  V.  44,  p.  171,  (1896). 

(158)  Tollens:  Untersuchungen  liber  Kohlenhydrate.  Die  Landwirtschaft- 
lichen Versuchs-Stationen,  V.  39,  p.  401,  (1891). 

(159)  Tollens  and  Glaubitz:  Uber  den  Pentosan-gehalt  verschiedener 
Materialien,  welche  zur  Ernahrung  dienen,  und  in  den  Garungs-Industrieen  ange. 


Nutrition  Investigations. 


373 


wendet  werden,  und  tiber  den  Verbleib  des  Pentosans  bei  den  Operationen,  welcher 
die  obigen  Materialien  unterworfen  werden.  Journal  fiir  Landwirthschaft,  V.  45, 
p.  97,  (1897). 

(160)  Tromp  De  Haas  and  Tollens:  Untersuchungen  iiber  die  Pectionstoffe. 
Zeitschrift  des  Vereins  fiir  die  Riibenzucker-Industrie  der  Deutschen  Reiches,  V. 
45,  p.  473,  (1895).    Liebig's  Annalen,  V.  286,  p.  278,  (1895). 

(161)  Utzjanzew:  Zur  Physiologie  des  Blinddarmes  bei  den  Pflanzenfressern. 
Biochemische  Zeitschrift,  V.  4,  p.  154,  (1907). 

(162)  Ward  and  Dunlop:  On  Some  Points  in  the  Histology  and  Physiology 
of  the  Fruits  and  Seeds  of  Rhamnus.    Annals  of  Botany,  V.  I,  p.  1,  (1887). 

(163)  Widtsoe  and  Tollens:  Uber  Arabinose,  Xylose  und  Fucose  aus 
Traganth.    Berichte  der  deutschen  chemischen  Gesellschaft,  V.  33,  p.  132,  (1900). 

(164)  Weiser:  tjber  die  Verdaulichkeit  der  Pentosane.  Die  Landwirt- 
schaftlichen  Versuchs-Stationen,  V.  58,  p.  238,  (1903). 

(165)  Weiser  and  Zaitschek:  Beitrage  zur  Methodik  der  Starkebestimmung 
und  zur  Kenntnis  der  Verdaulichkeit  der  Kohlenhydrate.  Pfliiger's  Archiv  fiir 
Physiologie,  V.  93,  p.  98,  (1903). 

(166)  Weiske  tjber  die  Verdaulichkeit  der  in  den  vegetabilischen  Futter- 
mitteln  enthaltenen  Pentosane.  Zeitschrift  fiir  physiologische  Chemie,  V.  20, 
p.  489,  (1895). 

(167)  Winterstein:  Uber  das  pflanzliche  Amyloid.  Zeitschrift  fiir  physiolo- 
gische Chemie,  V.  17,  p.  353,  (1893). 

(168)  Zuntz  and  Utzjanzew:  Zur  Bedeutung  des  Blinddarmes  fiir  die  Ver- 
dauung  beim  Kaninchen.  Archiv  fiir  Anatomie  und  Physiologie,  physiologische 
Abtheilung,   1905,  p.  403. 

IV.  GALACTANS. 

(169)  Bauer:  Ueber  den  aus  Agar-agar  entstehenden  Zucker,  iiber  eine  neue 
Saure  aus  der  Arabinose  nebst  dem  Versuch  einer  Classification  der  Gallertbilden- 
den  Kohlehydrate  nach  den  aus  ihnen  enstehenden  Zuckerarten.  Journal  fiir 
praktische  Chemie,  V.  30,  p.  36,  7,  (1884). 

(170)  Bauer:  Weitere  Untersuchungen  uber  Alimentare  Galaktosurie.  Zen- 
tralblatt  fiir  innere  Medizin,  1906,  p.  1176. 

(171)  Bente:  Ueber  anderweitige  Darstellung  von  Levulinsaure.  Berichte 
der  deutschen  chemischen  Gesellschaft,  V.  8,  p.  417,  (1875). 

(172)  Bente:  Zur  Darstellung  der  Levulinsaure  und  iiber  Carragheenzucker. 
Berichte  der  deutschen  chemischen  Gesellschaft,  V.  9,  p.  1157,  (1876). 

(173)  Bierry  and  Giaja  :  Digestion  des  mannanes  et  des  galactanes.  Comp- 
tes  Rendus,  V.  148,  No.  8,  p.  507,  (1909). 

(174)  Boureuelot  and  Herissey:  Germination  de  la  graine  de  Caroubier. 
Comptes  Rendus,  V.  129,  p.  614,  (1899). 

(175 J  Brasch:  Ueber  das  Verhalten  nicht-garungsfahiger  Kohlehydrate  im 
tierischen  Organismus  mit  Beriicksichtigung  des  Diabetes.  Zeitschrift  fiir  Biolo- 
gie,  V.  50,  p.  113,  (1907). 

(176)  Castoro:  Beitrage  zur  Kenntnis  der  Hemicellulosen.  Zeitschrift  fiir 
physiologische  Chemie,  V.  48,  p.  96,  (1905);  V.  49,  p.  96,  (1906). 

(177)  Cremer:  Ueber  das  Verhalten  einiger  Zuckerarten  im  tierischen  Organis- 
mus.   Zeitschrift  fur  Biologie,  V.  29,  p.  484,  (1892). 


374 


Mary  Dames  Swartz, 


(178)  Fluckinger  and  Mayer:  Neues  Repertorium  fur  Pharmacie,  1868, 
p.  350.    (Cited  by  Haedike,  Bauer  and  Tollens.) 

(179)  Goret:  Etude  chimique  et  physiologique  de  quelques  albumens  comes 
de  graines  de  Legumineuses.    These,  Paris,  1901.    (Cited  by  Herissey.) 

(180)  Greenish:  Untersuchung  von  Fucus  Amylaceous.  Berichte  der  deut- 
schen  chemischen  Gesellschaft,  V.  14,  p.  2253,  (1881). 

(181)  Greenish:  Die  Kohlehydrate  des  Fucus  Amylaceous.  Ibid.,  V.  5, 
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(182)  Gran:  Die  Hydrolyse  des  Agars  durch  ein  Enzym.  Centralblatt  fur 
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(183)  Gruss:  Studien  iiber  Reservecellulose.  Botanisches  Centralblatt,  V. 
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(184)  Gruss:  Ueber  den  Umsatz  bei  der  Keimung  der  Dattel.  Berichte  der 
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>  (188)  Hofmeister:  Ueber  Resorption  und  Assimilation  der  Nahrstoffe. 
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(190)  Konig  and  Bettels:  Die  Kohlenhydrate  der  Meeresalgen  und  daraus 
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(193)  Lohrisch:  Die  Ursachen  der  chronischen  habituellen  Obstipation  im 
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(195)  Mallevre:  Der  Einfluss  der  als  Gahrungsprodukt  der  Cellulose  gebil- 
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(198)  Munk:  Der  Einfluss  des  Glycerins,  der  fliichtigen  und  festen  Fettsauren 
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(209)  Schmidt,  A.:  Neue  Beobachtungen  zur  Erklarung  und  rationellen 
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(212)  Schulze:  Ueber  die  Zellwandbestandtheile  der  Cotyledonen  von  Lupi- 
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(215)  Schulze:  Ueber  das  Vorkommen  eines  unloslichen  Schleimsaure 
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(216)  Schulze:  Zur  Kenntnis  der  in  den  Leguminosensamen  enthaltenen 
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(217)  Schulze,  Steiger  and  Maxwell:  Zur  Chemie  der  Pflanzenzellmem- 
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376 


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(218)  Schulze  and  Castoro:  Beitrage  zur  Kenntnis  der  Hemicellulosen. 
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(219)  Schulze  and  Steiger:  Untersuchungen  uber  die  stickstofffreien  Reser- 
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(220)  Sebor:  Ueber  die  Kohlenhydrate  des  Carragheen-Moos.  Botanisches 
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(221)  Strauss:  Ueber  das  Vorkommen  eini  ger  Kohlehydratefermente  bei 
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(222)  Ulander:  Untersuchungen  iiber  die  Kohlenhydrate  der  Flechten. 
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(223)  Volt:  Ueber  die  Glykogenbildung  nach  Aufnahme  verschiedener 
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(224)  Volt:  Ueber  das  Verhalten  der  Galactose  beim  Diabetiker.  Zeit- 
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(225)  Von  Planta  and  Schulze:  Ueber  ein  neues  krystallisirbares  Kohle- 
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V.  MANNANS. 

(227)  Bertrand:  Sur  la  presence  de  la  mannocellulose  dans  le  tissu  ligneux 
des  plantes  gymnospermes.    Comptes  Rendus,  V.  129,  p.  1025,  (1899). 

(228)  Bierry  and  Giaja:  Sur  la  digestion  des  mannanes  et  des  galactanes. 
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(229)  Bierry  and  Giaja:  Digestion  des  mannanes  et  des  galactanes. 
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(230)  Brown  and  Morris:  On  the  Existence  of  a  Cellulose-dissolving  Enzyme 
(Cytohydrolyst)  in  the  Germinating  Seeds  of  the  Grasses.  Journal  of  the  Chemical 
Society,  London,  V.  57,  p.  497,  (1890). 

(231)  Bourquelot  and  Herissey:  Sur  la  composition  de  l'albumen  de  la 
graine  du  Phoenix  canariensis  et  sur  les  phenomenes  chimiques  qui  accompagnent 
la  germination  de  cette  graine.    Comptes  Rendus,  V.  133,  p.  302,  (1901). 

(232)  Bourquelot  and  Herissey:  Sur  la  composition  de  l'albumen  de  la 
graine  de  caroubier:  production  de  galactose  et  de  mannose  par  hydrolyse.  Ibid., 
V.  129,  p.  228,  (1899).  Sur  la  composition  de  la  graine  de  caroubier.  Ibid.,  V.  129, 
p.  391,  (1899). 

(233)  Bourquelot  and  Herissey:  Germination  de  la  graine  de  caroubier: 
production  de  mannose  par  un  ferment  soluble.    Ibid.,  V.  129,  p.  614,  (1899). 

(234)  Bourquelot  and  Herissey:  Sur  les  ferments  solubles  produits  pendant 
la  germination  par  les  graines  a  albumen  corne.  Comptes  Rendus,  V.  130,  p.  42, 
(1900). 

(235)  Bourquelot  and  Herissey:  Sur  l'individualite  de  laseminase,  ferment 
soluble  secrete  par  les  graines  de  legumineuses  a  albumen  corne  pendant  la  germina- 
tion.   Ibid.,  V.  130,  p.  340,  (1900). 


Nutrition  Investigations . 


377 


(236)  Bourquelot  and  Herissey:  Sur  la  mecanisme  de  la  saccharification 
dcs  mannanes  du  corrozo  par  la  seminase  de  la  luzerne.  Ibid.,  V.  136,  p.  404, 
(1903). 

(237)  Castoro:  Beitrage  zur  Kenntnis  der  Hcmicellulosen.  Zeitschrift  fiir 
physiologische  Chemie,  V.  49,  p.  96,  (1906). 

(238)  Cremer:  Verhalten  einiger  Zuckerarten  im  tierischen  Organismus. 
Zeitschrift  fur  Biologie,  V.  29,  p.  484,  (1892). 

(239)  Dillingham:  A  Contribution  to  the  History  of  the  Use  of  Bark  Bread. 
Bulletin  of  the  Bussey  Institution,  Vol.  Ill,  Part  V.  120,  (1906). 

(240)  Dubat:  Composition  des  hydrates  de  carbone  de  reserve  de  I'albumen 
des  graines  de  quelques  Liliacees  et  en  particulier  du  Petit  Haux.  Comptes  Rendus, 
V.  133,  p.  942  (1901). 

(241)  Effront:  Sur  une  nouvelle  enzyme  hydrolytique,  "la  caroubinase." 
Ibid.,  V.  125,  pp.  38  and  116,  (1897).  Sur  la  caroubinase.  Ibid.,  V.  125,  p.  309, 
(1897). 

(242)  Fischer  and  Hirschberger:  Ueber  Mannose.  Berichte  der  deutschen 
chemischen  Gesellschaft,  V.  21,  p.  1805,  (1888);  V.  22,  pp.  365  and  1155,  (1889). 

(243)  Franck:  Ueber  die  anatomische  Bedeutung  und  die  Enstehung  der 
vegetabilischen  Schleime.  Jahrbucher  fiir  wissenschaftliche  Botanik,  V.  5,  p.  161, 
(1866). 

(244)  Gans  and  Tollens:  Ueber  die  Bildung  von  Zuckersaure  aus  Dextrose 
haltenden  Stoffen,  besonders  aus  Rafhnose,  und  iiber  die  Untersuchung  einiger 
Pflanzenschleimarten.    Liebig's  Annalen,  V.  249,  p.  215,  (1888). 

(245)  Gatin:  Action  de  quelques  diastases  animales  sur  certaines  mannanes. 
Comptes  Rendus  de  la  Societe  de  Biologie,  V.  58,  p.  847,  (1905). 

(246)  Gatin  and  Gatin:  tiber  die  Verdaulichkeit  der  Mannanen  durch  Dias- 
tasen  der  hoheren  Tiere.    Chemisches  Centralblatt,  1907  (2),  p.  1181. 

(247)  Gatin:  Isomerisation  de  mannose  en  glycose  sous  Taction  d'un  fermenl 
soluble.    Comptes  Rendus  de  la  Societe  de  Biologie,  V.  64,  p.  903,  (1908). 

(248)  Girand:  Etude  comparative  des  gommes  et  des  mucilages.  Comptes 
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(249)  Goret:  Sur  la  composition  de  I'albumen  de  la  graine  de  fevier  d'Ameri- 
que  (Gleditschia  triacanthos  L.,  Legumineuses) .  Comptes  Rendus,  V.  131,  p.  60, 
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(250)  Gruss:  Studien  iiber  Reserve-Cellulose.  Botanisches  Centralblatt, 
V.  70,  p.  242,  (1897). 

(251)  Gruss:  Ueber  den  Umsatz  bei  der  Keimung  der  Dattel.  Berichte  der 
deutschen  botanischen  Gesellschaft,  V.  20,  p.  36,  (1902). 

(252)  Herissey:  Sur  la  digestion  de  la  mannane  des  tubercules  d'Orchidees. 
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(253)  Herissey:  Recherches  chimiques  et  physiologiques  sur  la  digestion  des 
mannanes  et  galactanes  par  la  seminase  chez  les  vegetaux.  Revue  Generale  de 
Botanique,  1903,  p.  345. 

(254)  Hilger:  Zur  Kenntnis  der  Pflanzenschleime.  Berichte  der  deutschen 
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(255)  Kano  and  Iishima:  Bulletin  of  the  College  of  Agriculture.  Tokyo  Im- 
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(256)  Kinoshita:  On  the  Occurrence  of  two  kinds  of  Mannan  in  the  Root  of 
Conophallus  Konjaku.  Bulletin  of  the  College  of  Agriculture,  Tokyo  Imperial 
University,  No.  V,  p.  205,  (1902). 

(257)  Kinoshita:  On  the  Occurrence  of  Mannan.  Ibid.,  No.  V,  p.  253, 
(1902). 

(258)  Meigen  and  Spreng:  Ueber  die  Kohlehydrate  der  Hefe.  Zeitschrift 
fur  physiologische  Chemie,  V.  55,  p.  48,  (1908). 

(259)  Mulder:  Ueber  Pflanzenschleim.  Journal  fur  praktische  Chemie,  V. 
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(260)  Neuberg  and  Mayer:  Schicksal  der  drei  Mannosen  im  Kaninchenleib. 
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(261)  Newcombe:  Cellulose  Enzymes.  Annals  of  Botany,  V.  13,  p.  49,  (1899). 

(262)  Niebling:  Untersuchungen  iiber  die  kiinstliche  Verdauung  landwirth- 
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(263)  Pohl:  Ueber  die  Fallbarkeit  colloider  Kohlenhydrate  durch  Salze. 
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(264)  Reiss:  Ueber  die  Natur  der  Reserve-Cellulose.  Berichte  der  deutschen 
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(265)  Rosenfeld:  Untersuchungen  iiber  Kohlehydrate.  Centralblatt  fur 
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(266)  Sachs:  Zur  Keimungsgeschichte  der  Dattel.  Botanische  Zeitung, 
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(267)  Sawamura:  On  the  Liquefaction  of  Mannan  by  Microbes.  Central- 
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(268)  Sawamura:  On  the  Digestive  Power  of  the  Intestinal  Canal.  Bulletin 
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(269)  Schellenberg  :  Untersuchungen  iiber  das  Verhalten  einiger  Pilze 
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(270)  Schmidt,  C:  Ueber  Pflanzenschleim  und  Bassorin.  Liebig's  Annalen 
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(271)  Storer:  Testing  for  Mannose.  Bulletin  of  the  Bussey  Institution, 
Vol.  Ill,  Part  II,  p.  13,  (1902). 

(272)  Storer:  Notes  on  the  Occurrence  of  Mannan  in  the  Wood  of  Some  Kinds 
of  Trees,  and  in  Various  Roots  and  Fruits.    Ibid.,  V.  Ill,  Part  III,  p.  47,  (1903). 

(273)  Schulze:  Zur  Chemie  der  pflanzlichen  Zellmembranen.  Zeitschrift  fur 
physiologische  Chemie,  V.  16,  p.  387,  (1892). 

(274)  Schuster  and  Liebscher:  Der  Nahrwerth  der  Steinnussspahne. 
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(275)  Strauss:  Ueber  das  Vorkommen  einiger  Kohlehydratefermente  bei 
Lepidopteren  und  Dipteren  in  verschiedenen  Entwicklungsstadien.  Zeitschrift  fur 
Biologie,  V.  52,  p.  95,  (1908). 

(276)  Thamm:  Ein  Beitrag  zur  Kenntnis  der  Pflanzenschleime.  Dissertation, 
Miinchen,  1903. 

(277)  Tollens  and  Gans:  Mannose  oder  Isomanitose  aus  Salepschleim. 
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(278)  Tollens  and  Oshima:  Uber  das  Nori  aus  Japan.  Berichte  der  deut- 
schen chemischen  Gesellschaft,  V.  34,  p.  1422,  (1901). 

(279)  Tollens  and  Widstoe  :  Uber  die  Reactionen  des  Methvl-Furfurols  und 
der  Methyl-Pentosane.    Ibid.,  V.  33,  p.  132,  (1900). 


Nutrition  Investigations. 


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(280)  Tsuji:  Mannan  as  an  Article  of  Human  Food.  Bulletin  of  the  College 
of  Agriculture,  Tokyo  Imperial  University,  No.  II,  p.  103,  (1894). 

(281)  Tsukamato:  Ueber  die  Bildung  von  Mannan  in  Amorphophallus 
Konjak.    Chemisches  Centralblatt,  V.  97a,  p.  930,  (1897). 

(282)  Van  Ekenstein:  Sur  la  caroubinose  et  sur  la  d-mannose.  Comptes 
Rendus,  V.  125,  p.  719,  (1897). 

(283)  Voit:  Ueber  die  Aufnahme  des  Pflanzenschleims  und  des  Gummis  aus 
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(284)  Weiske:  Versuche  iiber  die  Verdaulichkeit  und  den  Nahreffect  des 
Jobannisbrodes.    Journal  fur  Landwirthschaft,  V.  27,  p.  321,  (1879). 

(285)  Zanotti:  Untersuchungen  iiber  einige  zusammengesetzte  Kolhehy- 
drate.    Chemisches  Centralblatt,  V.  99a,  p.  1209,  (1899). 

VI.  LEVULANS. 

(286)  Bierry:  Recherches  sur  la  digestion  de  Pinuline.  Comptes  Rendus  de 
la  Societe  de  Biologie,  V.  59,  p.  256,  (1905). 

(287)  Bierry:  Recherches  sur  la  digestion  de  l'inuline.  Comptes  Rendus, 
V.  150,  p.  116,  (1910). 

(288)  Bierri  and  Porter:  Recherches  sur  la  digestion  de  l'inuline.  Ibid., 
V.  52,  p.  423,  (1900). 

(289)  Bourquelot:  Inulase  et  fermentation  alcoholique  indirecte  de  l'inuline. 
Comptes  Rendus,  V.  116,  p.  1143,  (1893). 

(290)  Bourquelot:  Remarques  sur  les  ferments  solubles  secretes  par  l'As- 
pergillus  et  le  Penicillium.  Comptes  Rendus  de  las  Societe  de  Biologie,  V.  9, 
p.  653,  (1893). 

(291)  Chevastelon:  Sur  l'inuline  d'ail,  de  la  jacinthe,  de  l'asphodele  et  de  la 
tub£reuse.    Journal  de  Pharmacie,  V.  4,  p.  2,  (1895).    (Cited  by  Dean.) 

(292)  Chittenden:  The  Behavior  of  Inulin  in  the  Gastro-intestinal  Tract. 
American  Journal  of  Physiology,  V.  2,  p.  XVII,  (1898). 

(293)  Dean:  Experimental  Studies  on  Inulase.  Botanical  Gazette,  V.  35, 
p.  24,  (1903). 

(294)  Dean:  On  Inulin.    American  Chemical  Journal,  V.  32,  p.  69,  (1904). 

(295)  Ducamp:  Beitrag  zum  Studium  der  Unterscheidung  des  Colibazillus, 
Wirkung  der  Bazillen  der  Colityphusruhrgruppe  auf  die  Kohlehydrate.  Jahres- 
bericht  fur  Thierchemie,  V.  37,  p.  952,  (1907). 

(296)  Ekstrand  and  Joh anson :  Zur  Kenntnis  der  Kohlehydrate,  I.  Berichte 
der  deutschen  chemischen  Gessellschaft,  V.  20,  p.  3310,  (1887).  Zur  Kenntnis  der 
Kohlehydrate,  II.    Ibid.,  V.  21,  p.  594,  (1888). 

(297)  Finn:  Experimentelle  Beitrage  zur  Glycogen-  und  Zuckerbildung  in  der 
Leber.  Arbeiten  aus  dem  physiologischen  Laboratorium,  Wiirzburg,  1877.  (Cited 
by  Miura.) 

(298)  Fitz:  Ueber  Schizomycetengahrungen.  Berichte  der  deutschen  chemi- 
schen Gesellschaft,  V.  11,  p.  42,  (1878). 

(299)  Frerichs:  Zur  Glykogenbildung  in  der  Leber.  Dissertation,  Wiirz- 
burg, 1876.    (Cited  by  Miura.) 

(300)  Green:  On  the  Germination  of  the  Jerusalem  Artichoke  (Helianthus 
tuberosus).    Annals  of  Botany,  V.  I,  p.  223,  (1888). 

(301)  Harlay:  De  l'hydrate  de  carbone  de  reserve  dans  les  tubercules  de 
'avoine  a  chapelets.    Comptes  Rendus,  V.  132,  p.  423,  (1901). 


380 


Mary  Dames  Swartz, 


(302)  Kiliani:   Ueber  Inulin.    Liebig's  Annalen,  V.  205,  p.  145,  (1880). 

(303)  Komanos:  Ueber  die  Verdauung  des  Inulins  und  seine  Verwendung 
bei  Diabetes  Mellitus.  Dissertation,  Strassburg,  1875.  Jahresbericht  fur  Thier- 
chemie,   V.6,  p.  180,  (1876). 

(304)  Kobert:  Ueber  einige  Enzyme  wirbelloser  Tiere.  Pfliiger's  Archiv  fur 
Physiologie,  V.  99,  p.  116,  (1903). 

(305)  Kulz:  Beitrage  zur  Pathologie  und  Therapie  des  Diabetes  Mellitus. 
Jahresbericht  fur  Thierchemie,  V.  4,  p.  448,  (1874). 

(306)  Kulz:  Beitrage  zur  Kenntnis  des  Glycogens.  Centralblatt  fur  Physio- 
logie, 1890,  p.  789. 

(307)  Levy:  De  la  fermentation  alcoholique  des  topinambours,  sous  l'influence 
des  levures.    Comptes  Rendus,  V.  116,  p.  1381,  (1893). 

(308)  Lindner:  Garversuche  mit  verschiedenen  Hefen  und  Zuckerarten. 
Chemisches  Centralblatt,  1901,  p.  56. 

(309)  v.  Lippmann:  Ueber  das  Lavulan,  eine  neue,  in  der  Melasse  der  Riiben- 
zucker-fabriken  vorkommende  Gummiart.  Berichte  der  deutschen  chemischen 
Gesellschaft,  V.  11,  p.  57.  (1881). 

(310)  Luchsinger:  Zur  Glykogenbildung  in  der  Leber.  Pfluger's  Archiv  fur 
Physiologie,  V.  8,  p.  289,  (1874). 

(311)  Mendel  and  Mitchell:  On  the  Utilization  of  Various  Carbohydrates 
without  Intervention  of  the  Alimentary  Digestive  Processes.  American  Journal 
of  Physiology,  V.  14,  p.  239,  (1905). 

'  (312)  Mendel  and  Nakaseko:  Glycogen  Formation  after  Inulin  Feeding. 
Ibid.,  V.  4,  p.  246,  (1900). 

(313)  Miura:  Wird  durch  Zufuhr  von  Inulin  die  Glykogenbildung  gesteigert? 
Zeitschrift  fur  Biologie,  V.  32,  p.  255,  (1895). 

(314)  Reidemeister:  Sinistrin,  Levulin,  und  Triticin.  Chemisches  Central- 
blatt, 1880,  p.  808;  Jahresbericht  fur  Thierchemie,  V.  11,  p.  68,  (1881). 

(315)  Richaud:  Sur  quelques  points  relatifs  a  l'histoire  physiologique  de 
l'inuline  chez  les  animaux.  Comptes  Rendus  de  la  Societe  de  Biologie,  V.  52,  p. 
416,  (1900). 

(316)  Saiki:  Anti-inulase.  Journal  of  Biological  Chemistry,  V.  3,  p.  395, 
(1907). 

(317)  Sandmeyer:  Ueber  die  Folgen  der  partiellen  Pancreasextirpation  beim 
Hund.    Zeitschrift  fur  Biologie,  V.  31,  p.  32,  (1895). 

(318)  Schmiedeberg:  Ueber  ein  neues  Kohlehydrat.  Zeitschrift  fur  physio- 
logische  Chemie,  V.  3,  p.  112,  (1879). 

(319)  Strauss:  Ueber  das  Vorkommen  einiger  Kohlehydrate  fermente  bei 
Lepidopteren  und  Dipteren.    Zeitschrift  fur  Biologie,  V.  52,  p.  95  (1908) . 

(320)  Tanret:  Sur  la  levulosane,  nouveau  principe  immediat  des  cereales 
Comptes  Rendus,  V.:   112,  p.  293,  (1891). 

(321)  Tanret:  Sur  l'inuline  et  deux  principes  immediats  nouveaux.  Ibid., 
V.  116,  p.  514,  (1893). 

(322)  Tanret:  Sur  les  hydrates  de  carbone  du  topinambour.  Ibid.,  V.  117, 
p.  50,  (1893). 

(323)  Tanret:  Sur  une  nouvelle  glucosane,  la  levoglucosane.  Ibid.,  V.  119, 
p.  158,  (1894). 

(324)  Von  Mering:  Zur  Glykogenbildung  in  der  Leber.  Pfluger's  Archiv 
fur  Physiologie  V.  14,  p.  274,  (1877). 


Nutrition  Investigations . 


381 


(325)  Wallach:  Zur  Kcnntnis  der  Kohlchydrate.  Liebig's  Annalen,  V.  234, 
p.  364,  (1886). 

(326)  Weinland:  Ueber  das  Auftreten  von  Invertin  im  Blut.  Zeitschrift 
fur  Biologie,  V.  47,  p.  280,  (1906). 

(327)  Went:  Monilia  sitophila  (Mont.)  Sacc,  ein  technischer  Pilz.  Che- 
misches  Centralblatt,  V.  (1901)  b,  p.  650. 

(328)  Winogradsky:  Clostridium  pastorianum,  seine  Morphologie  und  seine 
Eigenschaften  als  Buttersaureferment.  Chemisches  Centralblatt,  V.  1902,  b, 
p.  709. 

VII.  DEXTRANS. 

(329)  Bauer:  Ueber  eine  aus  Leinsamenschleim  entstehende  Zuckerart.  Die 
Landwirtschaftlichen  Versuchs-Stationen,  V.  40,  p.  480. 

(330)  Bauer:  Ueber  eine  aus  Laminariaschleim  entstehende  Zuckerart. 
Berichte  der  deutschen  chemischen  Gesellschaft,  V.  22,  p.  618,  (1899). 

(331)  Bauer:  Ueber  die  Arabonsaure  und  die  aus  Lichenin  entstehende 
Zuckerart.    Journal  fur  praktische   Chemie,  V.  34,  p.  46,  (1886). 

(332)  Berg:  Jahresbericht  fur  Chemie,  1873,  p.  848.  (From  Russ.  Zeitschr. 
Pharm.,  1873,  pp.  129  and  161.) 

(333)  Berzelius:  Recherches  sur  la  nature  du  lichen  d'Islande,  et  sur  son 
emploi  comme  aliment.    Annales  de  Chimie,  V.  90,  p.  277,  (1814). 

(334)  Brown:  Notes  on  Cetraria  Islandica.  American  Journal  of  Physiology, 
V.  I,  p.  455,  (1898). 

(335)  Escombe:  Chemie  der  Membranen  der  Flechten  und  Pilze.  Zeitschrift 
fur  physiologische  Chemie,  V.  22,  p.  288,  (1896). 

(336)  Honig  and  St.  Schubert:  Ueber  Lichenin.  Monatshefte  fur  Chemie, 
V.  8,  p.  452,  (1887). 

(337)  Klason:  tiber  die  durch  Inversion  von  Lichenin  entstehende  Zucker. 
Berichte  der  deutschen  chemischen  Gesellschaft,  V.  19,  p.  2541,  (1886). 

(338)  Meyer:  Ueber  Reservestoffe,  Kerne  und  Sporenbildung  der  Bakterien. 
Chemisches  Centralblatt,  V.  1900,  b.  p.  56,  (1900). 

(339)  Meigen  and  Spreng:  Ueber  die  Kohlehydrate  der  Hefe.  Zeitschrift 
fur  physiologische  Chemie,  V.  55,  p.  49,  (1908). 

(340)  Mendel:  Das  Verhalten  einiger  unverdaulicher  Kohlehydrate  im  Ver- 
dauungstrakt.  Zentralblatt  fur  die  gesammte  Physiologie  und  Pathologie  des 
Stoffwechsels,  No.  17,  p.  1,  (1908). 

(341)  Muller,  Karl:  Die  Chemische  Zusammensetzung  der  Zellmembranen 
bei  verschiedenen  Kryptogamen.  Zeitschrift  fur  physiologische  Chemise,  V. 
45,  p.  264,  (1905). 

(342)  Nilson:  Kenntnis  der  Kohlenhydrate  in  den  Flechten.  Upsala 
Lakareforenings  Forhandlingar,  V.  28.  (Jahresbericht  fiir  Thierchemie,  V.  23,  p. 
53,  (1893). 

(343)  O'Sullivan:  Amylam  in  Wheat,  Rye  and  Barley.  Chemical  News, 
V.  44,  p.  258,  (1881). 

(344)  Poulsson:  Untersuchungen  iiber  das  Verhalten  einiger  Flechten- 
kohlehydrate  im  menschlichen  Organismus  und  iiber  die  Anwendung  derselben 
bei  Diabetes-Mellitus.  Festschrift  fiir  Olof  Hammarsten  XIV,  Upsala  Lakarefore- 
nings Forhandlingar,  (1906). 


382  Mary  Dames  Swarlz. 

(345)  Rothentusser  :  Der  Schleimkorper  des  Leinsamens.  Dissertation, 
Miinchen,  1903.    (Jahresbericht  fur  Thierchemie,  V.  34,  p.  78,  1904. 

(346)  Saikj:  The  Digestibility  and  Utilization  of  Some  Polysaccharide  Carbo- 
hydrates derived  from  Lichens  and  Marine  Algae.  Journal  of  Biological  Chem- 
istry, V.  2,  p.  251,  (1906). 

(347)  Torup:  A  New  Carbohydrate  from  Laminaria  Digitata.  Biochemisches 
Centralblatt,  V.  8,  p.  770,  (1909).    (From  Pharmacia,  V.  6,  p.  153.) 

(348)  Ulander:  Untersuchungen  iiber  die  Kohlenhydrate  der  Flechten. 
Dissertation,  Gottingen,  (1905). 

(349)  Voit:  Hermann's  Handbuch  der  Physiologie,  V.  6,  p.  413,  (1881). 

(350)  Winterstein:  Zur  Kenntnis  der  in  den  Membranen  der  Pilze  enthal- 
tenen  Bestandtheile.  Zeitschrift  fur  physiologische  Chemie,  V.  19,  p.  521,  (1895). 
V.  21,  p.  152,  (1897). 

(351)  Von  Mering:  Zur  Glykogenbildung  in  der  Leber.  Pfliiger's  Archiv 
fiir.  Physiologie,  V.  14,  p.  274,  (1877). 

(352)  Yos,hihura:  Ueber  einige  Pflanzenschleime.  Jahresbericht  fiir  Thier- 
chemie, V.  25,  p.  51,  (1895).  (Bulletin  II,  No.  4,  College  of  Agriculture,  Tokyo 
Imperial  University.) 


A  CONSIDERATION  OF   SOME   CHEMICAL  TRANSFORMA- 
TIONS OF  PROTEINS  AND  THEIR  POSSIBLE  BEAR- 
ING ON  PROBLEMS  IN  PATHOLOGY* 


FRANK  P.  UNDERHILL 

NEW   HAVEN,  CONN. 

Recent  investigations  concerning  the  structure  of  proteins  have  led 
to  a  readjustment  of  our  ideas  with  respect  to  the  manner  in  which  these 
substances  may  become  an  integral  part  of  the  organism;  and  the  study 
of  the  changes  which  occur  from  the  time  when  protein  has  been  built 
up  into  cell  structure  until  its  exit  from  the  body  in  the  form  of  waste 
products  is  at  present  only  in  its  infancy.  According  to  the  newer 
conception,  the  protein  molecule  is  a  huge  complex  consisting  of  the 
union  of  a  large  number  of  simple  amino-acids.  This  conception  is  due 
largely  to  the  researches  of  Emil  Fischer,  who  has  succeeded  in  fastening 
together  various  combinations  of  amino-acids  in  such  a  manner  that  the 
resulting  compound  behaves  in  some  respects  like  certain  of  the  proteins. 
Some  of  these  substances,  called  polypeptids,  have  indeed  been  isolated 
from  the  decomposition  of  native  protein.  According  to  Fischer,  proteins 
are  merely  mixtures  of  complex  polypeptids  and  cannot  be  considered 
as  chemical  individuals.  This  view,  however,  is  not  shared  by  other 
protein  chemists. 

In  its  passage  through  the  alimentary  canal  the  large  protein  complex 
undergoes  a  degradation, — a  transformation  into  simpler  molecules  which 
are  still  regarded  as  simple  proteins,  together  with  a  long  chain  of  amino- 
acids  belonging  to  the  aliphatic,  aromatic  and  heterocyclic  series.  Our 
modern  view  of  the  nature  of  protein  forbids  the  acceptance  of  the  older 
idea  that  the  necessity  for  alimentary  treatment  of  protein  is  merely  to 
transform  protein  into  a  condition  suitable  for  absorption.  Obviously, 
solubility  and  diffusibility  are  only  a  small  portion  of  the  process ;  other- 
wise it  would  be  unnecessary  to  entail  so  much  labor  on  the  gastro-enteric 
tract  by  the  apparently  needless  formation  of  amino-acids.  The  need  for 
nitrogenous  substances  is  to  replace  cellular  structures  worn  out  through 
metabolic  activities.  Just  how  this  replacement  occurs  is  problematical. 
Certain  it  is  that  the  only  detectable  nitrogenous  food  supply  for  such 
structures  is  to  be  found  in  the  proteins  of  the  blood.  No  amino-acids 
or  other  protein  decomposition  products  have  been  isolated  from  the 

*  The  Middleton  Goldsmith  Lecture  for  1911;  read  before  the  New  York 
Pathological  Society  at  the  Academy  of  Medicine,  March  18,  1911. 


2 


blood.71' 3  An  explanation  for  the  necessity  for  the  extensive  disintegra- 
tion of  the  protein  molecule  has  been  offered  as  follows : 

"Every  species  of  animal — in  fact,  every  individual — has  its  specifically  con- 
stituted tissues  and  cells.  If  the  diet  were  always  the  same,  the  formation  of 
the  tissues  might  bear  some  close  relation  to  the  components  of  the  food.  The 
diet  varies,  however,  and,  especially  in  the  case  of  human  beings  and  the 
omnivora,  is  exceedingly  diverse  in  nature  and  to  make  its  organism  independent 
of  the  outer  world  in  the  matter  of  food  taken,  it  disintegrates  the  nutrient  it 
receives,  and  utilizes  those  components  which  may  be  of  service  to  it  in  building 
up  new  complexes."1 

Many  important  functions  have  been  attributed  to  the  intestinal  wall, 
but  perhaps  none  of  more  significance  than  a  selective  power  for  the 
synthesis  of  amino-acids  into  serum  albumins.  According  to  this  view, 
the  intestine  receives  a  series  of  more  or  less  simple  amino-acids  and  by 
uniting  them  in  varying  proportions  forms  definite  compounds,  probably 
serum  proteins,  to  meet  the  organism's  requirements.  The  serum  proteins 
are  then  drawn  on  to  furnish  nitrogen  requirements  for  the  specific 
organ  or  group  of  cells.  This  probably  entails  a  further  transforma- 
tion. Such  a  theory  accounts  for  the  necessity  of  the  complete  degrada- 
tion of  protein  in  the  gastro-enteric  canal  and  accords  with  the  entire 
absence  of  cleavage  products  in  the  blood.  On  the  other  hand,  this 
extensive  degradation,  synthesis  and  further  disintegration  appears  quite 
uneconomical  physiologically.  In  the  first  place,  only  a  comparatively 
small  amount  of  nitrogen  is  needed  to  rebuild  tissue,  and  the  excess  must 
be  transformed  into  some  form  easy  of  elimination,  all  of  which  entails 
loss  of  energy  and  tissue  waste.  It  is  a  matter  of  common  observation 
that  when  protein  is  fed,  practically  all  the  nitrogen  is  rapidly  excreted, 
and  one  is  inclined  strongly  to  believe  that  this  portion  has  never  been 
re-synthesized  into  protein.  If  amino-acids  or  other  decomposition 
products  could  be  found  in  the  blood,  the  solution  of  the  problem  would 
be  at  hand.  The  failure  to  discover  them  does  not  disprove  their  presence, 
however,  since  the  quantity  in  the  portal  vein,  at  any  one  moment,  may 
be  so  small  as  to  escape  detection  with  our  present  methods. 

Whichever  way  one  views  this  problem,  the  fact  remains  that  sooner 
or  later  intermediary  processes  must  be  concerned,  primarily  with  a 
series  of  amino-acids.  These  are  the  substances  that  must  be  metabolized 
in  either  case,  and  it  is  to  some  of  the  transformations  that  these  com- 
pounds may  undergo  within  the  organism  that  I  particularly  desire  to 
call  attention.  It  is  my  purpose  to  indicate  the  possible  bearing  of  certain 
types  of  intermediary  processes  upon  problems  in  pathology,  for  it  is  my 
belief  that  the  hope  for  a  complete  understanding  of  some  of  the  phases 
of  disease  will  be  realized  in  proportion  as  our  knowledge  of  intermediary 
processes  increases. 


3 


DEAMINATION  AND  THE  SIGNIFICANCE  OF  AMMONIA  IN  THE  BODY 

Modem  investigations  teach  that  when  amino-acids  obtain  entrance 
to  the  tissues,  a  process  of  deamination  rapidly  occurs;  the  nitrogen  is 
split  off  in  the  form  of  ammonia.  In  other  words,  the  amino-acid  is 
converted  into  a  nitrogenous  and  a  non-nitrogenous  portion.  It  is 
probable  that  this  process  of  deamination  takes  place  for  the  most  part 
in  the  liver,  although  the  liver  is  by  no  means  the  only  organ  capable 
of  performing  this  reaction,  as  has  been  pointed  out  by  Jacoby51  and 
Lang.57  The  older  observation  of  Nenki,  that  blood  flowing  from  the 
intestine  is  richer  in  ammonia  than  the  blood  of  any  other  vessel,  may 
also  be  regarded  as  probably  our  first  indication  that  the  intestinal  wall 
may  carry  out  the  process  of  deamination.  Ordinarily,  the  ammonia 
split  off  reappears  in  the  urine  as  urea,  being  synthesized  into  this  closely 
related  compound  in  the  liver.  That  deamination  occurs  also  with  certain 
protein  complexes,  has  been  conclusively  demonstrated  by  Cohnheim.24 
The  further  transformation  of  the  non-nitrogenous  part  will  be  consid- 
ered later. 

Under  normal  conditions  the  quantity  of  ammonia  excreted  in  the 
urine  varies  within  certain  well  defined  limits,  and  in  general  is  directly 
proportional  to  the  intake  and  total  output  of  nitrogen.  The  elimination 
of  ammonia  in  disease  may  vary  enormously,  and  usually  when  a  changed 
excretion  is  observed  it  is  in  the  direction  of  a  greatly  increased  output. 
In  fact,  ammonia  output  in  the  urine  is  probably  more  easily  affected 
than  the  elimination  of  any  other  single  urinary  constituent,  except  the 
excretion  of  urea  which  bears  a  reciprocal  relation  to  ammonia.  If  the 
literature  relating  to  ammonia  output  in  disease  is  reviewed,  it  is  found 
that  increased  ammonia  excretion77  is  characteristic  of  pathological  con- 
ditions apparently  widely  diverse  in  nature.  For  instance,  an  augmented 
ammonia  output  has  been  observed  in  cholera,  intestinal  hepatitis, 
carcinoma  of  the  liver,  cirrhosis  of  the  liver,  pneumonia,  polyarthritis, 
typhoid,  various  other  fevers,  acute  uremia,  phosphorus  poisoning, 
gastro-enteritis,  starvation,  diabetes,  pernicious  vomiting  of  pregnancy, 
eclampsia,  etc. 

Ammonia  in  the  normal  urine  is  looked  on  to-day  as  an  index  to 
the  quantity  of  acids  present.  Acids  are  toxic  to  the  organism,  as  has 
been  demonstrated  repeatedly,  and  ammonia  is  diverted  from  its  trans- 
formation into  urea  to  neutralize  these  acid  radicles.42'  44»  25>  31> 52  The 
same  idea  prevails  with  respect  to  diseased  conditions  in  which  increased 
ammonia  in  the  blood  and  urine  is  regarded  as  evidence  of  increased 
production  of  acid  radicles.77  It  is  a  well-known  observation  that  this 
excessive  elimination  may  be  greatly  diminished  by  feeding  other  alkalies, 
as,  for  instance,  in  diabetes.  If  ammonia  is  employed  in  the  tissues 
merely  to  neutralize  acid  radicles,  it  would  seem  fair  to  assume  that  if 


4 


sufficient  alkali  were  introduced,  no  ammonia  should  appear  in  the  urine. 
Experimentally,  such  a  condition  has  never  been  brought  about  in  spite 
of  very  large  doses  of  alkali.  From  this  fact  it  would  appear  that  a 
certain  quantity  of  ammonia  is  continually  present  in  the  blood.  This 
holds  true  also  for  the  herbivora,  even  though  these  animals  are  assumed 
to  neutralize  acid  radicles  by  alkalies  other  than  ammonia. 

Throughout  the  literature  relating  to  acidosis  and  ammonia  excretion 
in  the  urine,  emphasis  is  laid  on  acid  radicles  as  toxic  agents.  Little 
or  no  attention  has  been  devoted  to  the  role  which  may  be  played  by 
ammonia  itself  when  viewed  from  the  same  standpoint.  As  a  matter 
of  fact,  ammonia  is  exceedingly  poisonous  and  few  salts  exceed  those  of 
the  ammonium  series  in  comparative  toxicity.  On  the  other  hand,  of 
the  acid  radicles  which  are  supposed  to  exert  such  deleterious  effects, 
as,  for  example,  in  diabetes,  not  one  has  been  shown  to  have  any  strikingly 
toxic  properties,  at  least  in  the  normal  organism.  From  these  statements 
the  possibility  is  offered  that  increased  ammonia  output  in  the  urine  may 
really  mean  increased  ammonia  production  resulting  from  decreased 
urea  formation.  Experimentally,  there  is  no  evidence  that  such  may 
not  be  the  case,  and  according  to  this  view  ammonia  must  be  neutralized, 
which  would  account  for  the  presence  of  organic  acids  in  the  urine,  as 
in  diabetes,  starvation,  liver  disorders,  etc.,  precisely  as  the  introduction 
of  camphor,  menthol,  thymol,  etc.,  accounts  for  the  appearance  of 
glycuronic  acid  in  the  urine.  In  this  connection  another  interesting 
problem  presents  itself.  In  diabetes,  beta-oxybutric  acid  is  the  acid 
formed  in  large  quantity,  whereas  in  other  conditions,  as  in  cirrhosis  of 
the  liver,  lactic  acid  is  found.  As  a  rule,  the  two  do  not  occur  together 
except  in  starvation  and  in  pathological  conditions  in  which  inanition  is 
an  accompaniment.  The  presence  of  these  two  compounds  in  the  urine 
would  indicate  two  different  types  of  mechanism  slightly  diverted  from 
the  normal.93  In  entire  accord  with  the  theory  that  ammonia  may  be 
the  toxic  agent  are  the  observations  of  Carlson21  and  Jacobson.49  The 
former  has  shown  an  increased  quantity  of  ammonia  in  the  blood  after 
complete  thyroidectomy  and  parathyroidectomy.  The  latter  has  demon- 
strated that  the  concentration  of  ammonia  in  the  blood  necessary  to 
produce  experimental  ammonia  tetany  is  practically  equal  to  that  found 
during  parathyroid  tetany.  "This  supports  the  view  that  the  increased 
ammonia  in  the  blood  of  parathyroidectomized  animals  is  directly  respon- 
sible for  the  tetany  and  the  depressive  symptoms."49 

If,  on  the  assumption  that  a  common  law  underlies  all  the  various 
pathological  conditions  mentioned,  one  attempts  to  account  for  the  pres- 
ence of  ammonia  in  the  urine,  the  suggestion  presents  itself  that  it  is  in 
some  way  connected  with  the  metabolism  of  the  carbohydrates.  In  practi- 
cally every  condition  in  which  ammonia  in  the  urine  is  characteristic, 


5 


there  is  either  a  lack  of  carbohydrates,  as  in  prolonged  starvation,  or  a 
faulty  utilization  of  carbohydrate,  as  in  diabetes,  cirrhosis  of  the  liver, 
or  after  complete  removal  of  the  thyroids  and  parathyroids,  etc.92  It 
would  appear,  therefore  that  carbohydrate  is  the  factor  controlling  the 
regulation  of  ammonia  production.93  Thus  in  prolonged  starvation, 
ammonia  in  the  urine  may  be  greatly  diminished  by  feeding  carbo- 
hydrate.77' 93  In  the  solution  of  the  problem  as  to  the  part  played  by 
carbohydrate,  at  least  two  possibilities  are  presented:  1.  Carbohydrate 
influences  the  output  of  ammonia  indirectly  by  its  effect  on  the  combus- 
tion of  fat,  for  it  is  well  known  that  fat  is  burned  much  more  readily 
when  carbohydrate  is  present  than  when  the  store  of  this  substance  in  the 
body  is  greatly  depleted.  2.  It  may  be  possible  that  the  liver  cell,  for 
instance,  is  incapable  of  effecting  urea  synthesis  in  the  presence  of  insuf- 
ficient carbohydrate  in  that  organ.  The  latter  hypothesis  certainly  pre- 
sents interesting  problems,  both  for  the  physiologist  and  for  the  patholo- 
gist. Attempts  have  been  made  in  our  laboratory  to  follow  this  hypothesis 
to  its  logical  conclusion.  The  results  thus  far  obtained,  however,  have  not 
been  far-reaching  for  the  reason  that  it  is  exceedingly  difficult  to  obtain 
the  proper  experimental  conditions.  The  investigation  has  been  carried 
out  on  animals  entirely.  In  the  first  place,  an  endeavor  has  been  made 
to  remove  the  carbohydrate  store  from  the  animal's  body  by  various  means 
with  the  hope  of  inducing  increased  ammonia  in  the  urine.  Prolonged 
starvation,  phosphorus  poisoning,  a  combination  of  the  two,  phloridzin 
intoxication  alone  and  together  with  prolonged  inanition,  and  cocain 
poisoning  have  proved  unsuccessful  in  inducing  increased  output  of 
ammonia.  This  result  is  in  harmony  with  those  obtained  by  other  inves- 
tigators (Jackson  and  Pearce,48  Eichards  and  Wallace,83  Underhill  and 
Kleiner94).  These  facts  by  no  means  invalidate  the  hypothesis,  for  it  is 
well  known  from  the  researches  of  Pniiger  and  his  pupils  that  it  is 
exceedingly  difficult  to  get  dogs  glycogen-free.  Since  it  is  well  known 
that  dogs  are  refractory  in  this  respect,  inanition  experiments  were  car- 
ried out  also  with  the  pig  and  rat,  omnivora,  and  hence,  presumably, 
allied  to  man  in  metabolism.  The  results  were  in  entire  accord  with  those 
with  the  dog.  Ammonia  in  the  urine  was  not  increased.  From  this 
point  of  view  it  seems  most  probable  that  the  decision  of  this  question  can 
be  obtained  most  readily  with  the  human  subject. 

If  ammonia  is  to  be  regarded  merely  as  an  alkali  for  neutralization 
purposes,  rather  than  as  a  toxic  agent  per  se,  it  may  be  cited  as  a  splendid 
illustration  of  the  "factor  of  safety"  principle  enunciated  by  Meltzer67 
for  other  mechanisms.  Ordinarily,  ammonia  is  transformed  to  urea,  but 
if  acid  radicles  are  floating  in  the  body  fluids  they  are  rendered  inert  by 
union  with  ammonia,  hence  less  urea  is  formed.  Therefore,  increased 
ammonia  in  the  urine,  with  a  corresponding  diminution  in  the  urea  may 


6 


be  regarded  as  an  indication  that  the  body  is  endeavoring  to  maintain 
normal  conditions  and  not  that  there  is  necessarily  any  inability  to  form 
nrea  on  the  part  of  the  organism,  or  that  lesions  in  one  or  another  organ 
are  necessarily  related  to  increased  ammonia  output.  Indeed,  even  when 
acute  yellow  atrophy  of  the  liver  is  at  its  height,  the  quantity  of  ammonia 
in  the  urine  may  be  normal.77 

Closely  associated  with  problems  concerning  the  significance  of 
ammonia,  are  those  having  to  do  with  the  relation  of  this  substance  to  the 
amino-acids  and  the  mechanism  of  deamination.  Within  the  last  few 
years  discussion  of  the  relation  of  so-called  defective  or  insufficient  deami- 
nation in  a  series  of  pathological  conditions  has  come  into  vogue.  It  has 
been  assumed  that  the  liver  was  incapable  of  performing  its  function 
and  that  the  condition  was  indicated  by  high  ammonia  and  a  high  unde- 
termined nitrogen  in  the  urine.  It  does  not  seem  to  have  occurred  to 
those  who  advocate  this  point  of  view  that  high  ammonia  and  the  pres- 
ence of  amino-acids  in  the  urine  present  incongruous  conceptions  if  the 
sum  of  urea  and  ammonia  nitrogen  is  normal.  In  other  words,  if  deami- 
nation were  defective,  one  would  expect  low  ammonia  in  the  urine  when 
amino-acids  are  present.  Much  stress  has  been  laid  latterly  on  the  occur- 
rence of  amino-acids  in  the  urine.  In  health  a  little  glycocoll  has  been 
isolated  in  the  urine,  which  is  not  strange  when  one  considers  the  role 
of  glycocoll  as  a  compound  intended  to  render  toxic  substances  innocuous 
as  in  the  case  of  benzoic  acid,  and  the  comparative  difficulty  with  which 
glycocoll  is  burned.  Leucin  and  tyrosin  have  been  isolated  from  the 
urine  of  patients  with  acute  yellow  atrophy,  and  with  phosphorus 
poisoning.75 

According  to  Ignatowski,47>  4>  33>  34>  104>  88>  41-  65> 78  glycocoll,  leucin, 
tyrosin  and  aspartic  acid  are  found  in  pneumonia  and  leukemia.  Similar 
finds  are  obtained  in  scarlatina  and  typhoid.50' 39  Insults  to  the  pan- 
creas17 may  lead  to  the  excretion  of  a  polypeptid  which  on  hydrolysis 
yields  tyrosin,  and  according  to  several  observers70' 2  a  similar  compound 
may  be  obtained  from  diabetic  urines.  As  a  result  of  disturbed  meta- 
bolism induced  by  lack  of  oxygen,61' 62  amino-acids  may  appear  in  the 
urine  as  well  as  after  ether-chloroform  narcosis.5  These  bodies  may  also 
be  present  in  exudates  and  in  the  fluids  formed  in  edema.76' 75 

To  account  for  the  presence  of  amino-acids  in  the  blood  and  hence 
also  their  excretion  by  the  kidney,  it  is  unnecessary  to  assume  that  defec- 
tive deamination  is  responsible.  They  may  be  produced,  as  for  example, 
by  increased  autolysis  of  a  tissue  or  organ  in  a  part  of  the  body  far  remote 
from  the  organ  or  organs  possessing  the  power  of  deamination  and  may 
therefore  be  eliminated  through  the  urine  before  the  organs  of  deamina- 
tion have  had  an  opportunity  of  performing  their  function.  In  other 
words,  these  compounds  may  be  formed  in  the  muscles,  for  instance,  and 
be  eliminated  in  a  large  part  by  the  kidney  before  the  liver,  which  may  be 


7 


cited  as  an  organ  of  examination,  has  had  an  opportunity  of  acting  on 
them.  In  harmony  with  this  idea  may  be  cited  the  conditions  which 
obtain  in  this  respect  in  cystinuria.75  In  certain  of  these  patients,  the 
amino-acid,  cystin,  is  eliminated  through  the  urine  either  alone  or  in 
company  with  other  amino-acids,  for  example,  leucin  and  tyrosin.  Appar- 
ently, the  cystinuric  patient  is  incapable  of  deaminating  and  burning  the 
cystin.  Nevertheless,  when  cystin11' 102  is  fed  to  such  individuals  they 
experience  no  difficulty  in  completely  deaminating  and  burning  this 
amino-acid,  and  it  has  also  been  demonstrated  that  certain,  at  least  of  the 
cystinurics,  can  handle  other  amino-acids,  as  tyrosin90  and  aspartic  acid,103 
when  fed.  The  reason  for  the  presence  of  amino-acids  in  the  urine  under 
the  pathological  conditions  previously  cited,  may  be  similar  to  that  fre- 
quently given  for  cystinuria,  namely,  that  the  cystin  eliminated  arises  as  a 
result  of  processes  within  the  organism,  not  necessarily  from  the  food. 
And  in  neither  case  is  it  necesary  to  assume  defective  deamination. 


Until  recently  our  knowledge  concerning  the  exact  mode  of  decompo- 
sition of  the  cleavage  products  of  protein  has  been  extremely  limited.  It 
has  been  assumed  for  years  that  the  non-nitrogenous  portion  of  the  amino- 
acid  is  oxidized  and  hence  may  be  looked  on  either  as  a  direct  source  of 
energy,  or  as  potential  energy  residing  in  carbohydrates  or  fats  synthesized 
from  such  material.  Eecent  observations,  however,  have  tended  to  give 
us  a  more  enlightened  view  as  to  the  exact  way  in  which  these  amino- 
acids  are  handled.  Thus,  from  the  investigations  of  Emden,  Salomon  and 
Schmidt,35  it  may  be  seen  that  leucin  added  to  the  blood  of  a  dog  and 
made  to  pass  through  the  surviving  liver  causes  a  considerable  increase  in 
the  acetone  contained  in  the  circulating  blood.  Under  normal  conditions 
some  acetone  is  a  product  of  liver  activity.53 

In  order  to  understand  the  steps  necessary  for  the  production  of 
acetone  from  leucin,  certain  other  facts  must  be  taken  into  consideration. 
Knoop53  has  conclusively  demonstrated  that  the  aromatic  fatty  acids  are 
decomposed  in  the  body  in  such  way  that  there  is  an  oxidation  at  the 
beta  carbon  atom  followed  by  a  cleavage  in  the  side  chain  between  the 
alpha  and  beta  carbon  atoms.  Thus,  phenylbutyric  acid  is  decomposed 
into  acetic  acid  and  phenylacetic  acid.  The  latter  appears  in  the  urine, 
and  the  acetic  acid  is  presumably  decomposed  to  C02  and  H20. 


THE  FURTHER  FATE  OF  AMINO-ACIDS 


COOH 

phenyl  butyric  ucd 


8 


It  is  perfectly  possible  that  the  breaking  down  of  the  aliphatic  fatty 

acids  takes  place  in  somewhat  the  same  fashion.  Eeasoning  from  this 
viewpoint  it  is  probable  that  the  transformations  which  occur  in  the 
production  of  acetone  from  leucin  are  as  follows : 

CH3      CH,            CH,      CH3           CHZ       CH3  CH3  CH, 

\/7var1v.C0\/  \/ 

anamination         ^       c»eava5e  r>  r- /■» -f\ 


CH 


CH  C'eaW5e    .  PCH   k  C=0 


CH2  CHZ 

CH  NH2  C=0  COOH 

COOH  [too]" 

acid  with 

ammo    acid  ketone  acid  /€SS  cai-bon 


one 


(Leucn)  (Isovaleric  add)  (Ac*ton%) 

Leucin  first  undergoes  oxidative  deamination  by  which  a  ketone  acid 
is  formed.  Then  by  oxidative  C02  cleavage  isovaleric  acid  is  produced. 
Next  cleavage  takes  place  between  the  alpha  and  beta  carbon  atoms. 

On  the  other  hand,  Baer  and  Blum12  have  found  an  increased  output 
of  beta  oxybutyric  acid  in  the  urine  after  giving  leucin  to  a  diabetic  and 
the  following  transformations  have  been  attributed  to  it. 

CH  CH  CH  CHOH 

II  I  I 

CH2   >     CH,   >  CHo   *  CH0 

J  I  J  \ 

CH.NH2        CHOH  COOH  COOH 
L 


CH3      CH,  CH3      CH3      CH3    <CH3  CH3 


I 

COOH 


CocjH  @  oxybutyrio 

Leucin    Oxyisobutyl-      Isovaleric  acid 
acstic  acid  acid. 

Whether  one  can  assume  from  these  observations  that  the  organism 
of  the  diabetic  is  incapable  of  handling  the  amino-acid  in  a  normal  man- 
ner remains  a  problem  for  future  investigation  to  decide.  ''With  the 
proof  of  acetone  formation  from  products  obtained  from  protein  deriva- 
tives, we  obtain  for  the  first  time  a  clear  idea  concerning  the  utilization 
of  the  carbon  chains  free  from  nitrogen  from  certain  amino-acid s.  We 
learn  in  this  way  to  consider  the  formation  of  acetone  as  a  normal 
process,  it  being  a  normal  product  in  the  decomposition  of  leucin."1  If 
acetone  is  a  step  in  the  demolition  of  certain  of  the  amino-acids,  why  is  it 
that  in  various  pathological  conditions,  as  diabetes,  gastro-enteritis,  star- 
vation, etc.,  large  quantities  of  this  substance  are  excreted  in  the  urine 
instead  of  being  further  decomposed  as  in  health?  In  answer  to  this 
query,  it  may  be  stated  that  probably  only  a  small  portion  of  the  acetone 
excreted  under  the  conditions  just  cited  really  has  an  origin  in  the  carbon- 
free  rest  of  the  amino-acids. 


9 


When  a  fatty  acid  attached  to  the  benzene  ring  is  followed  through  the 
organism,  the  changes  which  occur  are  not  quite  so  simple.1' 20  Ordi- 
narily, when  fed  to  the  normal  organism,  tyrosin,  which  is  such  a  combi- 
nation, entirely  fails  of  detection  in  the  excreta.  Not  only  has  the  fatty 
acid  been  decomposed,  but  the  benzene  ring  has  been  broken,  which  may 
be  regarded  as  a  difficult  chemical  reaction.  The  possible  steps  through 
which  tyrosin  must  be  carried  have  been  indicated  by  Neubauer.74  There 
is  a  class  of  pathological  subjects  that  present  certain  anomalies  in  meta- 
bolism. These  subjects,  alkaptonurics,  excrete  urine  containing  homo- 
gentisic  acid.75  For  a  long  time  it  has  been  recognized  that  tyrosin  fed 
to  the  alkaptonuric  results  in  an  increased  output  of  homogentisic  acid. 
Ordinarily,  this  substance  is  decomposed  in  the  organism,  but  in  the  class 
of  individuals  referred  to,  it  would  appear  that  this  decomposition  is 
withheld  or  inhibited.  The  changes  which  lead  to  the  formation  of 
homogentisic  acid  in  the  alkaptonuric  and  hence  probably  also  in  the 
normal  subject  have  been  outlined  as  follows: 
OH 


nidation  %  y/Koxidative 
rearrange-  HO  /  \  CG-  c/e«v< 


HO 

wage 


p.oxypkcnyl  ^ 
amino  -  propionic 
a-cld  (Tyrosin) 


p.  oxypneny* 
pyroracemic  acid 
(^Ketone  acic 


rl 

Id) 


CH, 
I  2 

I 

|  CQO\H 

hydro      in  one 
pyroracemic 
acid 


Acetone 
bodies 


OH 


ho  m  o£  entis  /'c 
acid 


In  harmony  with  the  idea  that  homogentisic  acid  is  an  intermediary 
product  in  the  decomposition  of  tyrosin  is  the  demonstration  that  the 
normal  liver  is  capable  of  forming  acetone  from  homogentisic  acid.35 
The  reactions  which  occur  are  probably  in  accord  with  the  suggestion 
given  below : 


c 
i 

CH 


Homogentisic  acid 
Derivative  oftaiito- 


^  meric  form. 


(  a  ) 

(  b  ) 

(  c  ) 

CH2 

CH3 

CH3 

If 

I 

CH 

CH.OH 

c=o 

1 

1 

1 

CH2 
1 

CH, 
I 

CH2 
1 

COOH 

COOH 

COOH 

COOH 


crotonic  acid 


ft  oxyoutyric  diacetic 
acid  acid 


10 


So  far  as  the  condition  of  alkaptonuria  itself  is  concerned,  it  can 
hardly  be  looked  on  as  distinctly  a  pathological  state;  it  is  rather  an 
anomaly  of  metabolism.  Nevertheless,  it  is  possible  that  it  may  bear  a 
relation  to  disease.  Thus,  it  has  been  suggested  that  there  is  an  as  yet 
unexplained  relationship  between  alkaptonuria  and  chronic  polyar- 
teritis.32' 58» 79' 74  Homogentisic  acid  is  also  closely  related  to  certain  pig- 
ments which  occur  under  abnormal  conditions.  It  may  be  easily  trans- 
formed into  a  dark-colored  pigment  which  bears  a  relation  to  the  group 
of  bodies  known  as  melanins  which  are  also  protein  derivatives.  Whether 
homogentisic  acid  is  concerned  in  the  production  of  color  in  melanotic 
tumors,  remains  a  question  for  future  decision.  It  has  been  suggested10 
that  there  is  relationship  between  homogentisic  acid  and  the  coloring  of 
the  bones  in  ochronosis  and  at  times  ochronosis  and  alkaptonuria  occur 
simultaneously.59 

If  it  can  be  demonstrated  that  alkaptonuria  bears  a  direct  relationship 
to  certain  diseases,  it  is  obvious  that  the  complete  understanding  of  the 
chemical  transformations  which  lead  to  the  production  of  this  anomaly 
will  undoubtedly  prove  of  value  in  the  unraveling  of  other  abnormal 
processes. 

According  to  the  views  which  have  just  been  expressed  concerning  the 
fate  of  amino-acids  in  the  body,  the  non-nitrogenous  portion  is  merely 
broken  down  into  simpler  compounds  and  eliminated.  This  process  of 
degradation  undoubtedly  furnishes  a  certain  amount  of  energy  in  the 
form  of  heat.  Looked  at  from  another  point  of  view,64  the  non-nitrog- 
enous part  of  certain,  at  least,  of  the  amino-acids  may  be  synthesized  to 
carbohydrate  and  fat  which  in  turn  are  utilized  as  sources  of  energy. 

Thus  far,  the  whole  question  as  to  the  fate  of  amino-acids  in  the  body 
has  been  looked  on  as  one  of  analysis  or  demolition.  Eecently  it  has  been 
suggested  that  there  may  be  a  synthesis  of  amino-acids  within  the 
organism,  and  Knoop54  has  placed  on  record  certain  experiments  which 
tend  to  substantiate  this  view.  Thus,  after  feeding  phenyl  ketonebutyric 
acid  or  even  oxyacids  of  a  similar  type,  he  was  able  to  separate  from  the 
urine  an  acetyl  derivative  of  an  amino-acid.  Here,  for  the  first  time,  there 
is  established  a  direct  relationship  between  protein  and  carbohydrate 
metabolism. 

BACTERIAL  PRODUCTS 

Indol,  Skatol,  etc. — As  a  result  of  the  bacterial  digestion  of  protein, 
certain  well-known  and  well-defined  products  have  been  isolated  and  their 
specific  influence  on  the  organism  noted.  Not  only  have  these  bodies 
been  separated  and  identified  but  the  chemical  transformations  leading 
to  their  formation  have  been  made  clear.  The  protein  derivatives, 
tyrosin  and  tryptophan,  are  the  mother  substances  for  the  best-known 
bacterial  products.   In  the  intestinal  contents  have  been  found  a  number 


11 


of  substances  which  are  undoubtedly  derived  from  tyrosin.  These  com- 
pounds are:  OH  OH  oh  oh  QH 


CH2 

CH. NH2 
I 

COOH 

p.OXypAerty/   a  amino 


CH2 
I 

CH2 
I 

COOH 


CLC'CL  1  71 


y-os/nj  proftioht'c  acid 


C00H  c,«.l 

f>.  OXypnenyl 
<*C«fi'c  acid. 


According  to  these  reactions,  a  deamination  of  the  aromatic  amino- 
acid  must  occur  in  the  large  intestine.  The  further  reaction  for  the  pro- 
duction of  p.  oxyphenylacetic  acid  involves  the  cleavage  of  carbon 
dioxid  from  the  carboxylic  group  and  subsequent  oxidation  of  the  carbon 
atom.  If  phenol  is  produced  from  cresol,  then  demethylation  must  occur. 
These  reactions  if  correct,  must  involve  the  processes  of  deamination, 
cleavage  of  carbon  dioxid,  oxidation  and  demethylation.  From  trypto- 
phan the  following  products  are  formed. 


C.CHXH2.C00H 


CCH2.CH2.C00H 


CXH2.COOH 


C.CH, 


/?  amino 
tic  acid 


Indol  pt-opionic. 
Acid 


Xtidol  acetic 
acieC 


Sfcatol 


From  these  reactions  it  is  obvious  that  the  same  chemical  changes 
have  occurred  as  in  the  transformations  for  tyrosin,  namely,  deamination, 
carbon  dioxid  cleavage,  oxidation  and  finally  demethylation.  On  the 
other  hand,  it  has  been  suggested  recently  that  indol  may  arise  in  part  as 
a  result  of  intermediary  processes  quite  distinct  from  those  involved  in 
putrefaction.16 

Ordinarily,  when  intestinal  putrefaction  is  mentioned  one  invariably 
thinks  of  indol  and  skatol  as  being  responsible  for  the  series  of  disturb- 
ances which  may  be  associated  with  this  condition.  It  has  been  assumed 
that  a  long  list  of  pathological  conditions  may  be  closely  related  to 
increased  intestinal  putrefaction.  Thus,  this  condition  has  been  held 
responsible  in  part  for  sciatica,  tetany,  epilepsy,  eclampsia,  many  forms 
of  dermatitis,  cirrhosis,  arteriosclerosis,  various  types  of  nervous  diseases, 
chlorosis,  myxedema,  cretinism,  pernicious  anemia  and  nephritis.98' 73 


12 


Although  there  is  abundant  clinical  evidence  that  excessive  intestinal 
putrefaction  may  be  associated  with  or  responsible  for  marked  disturb- 
ances, the  substances  thus  far  isolated  from  intestinal  contents  can  not 
be  said  to  possess  very  profound  toxicity.  It  is  true  that  indol  when 
administered  in  quantities  up  to  2  gm.  per  day  causes  frontal  headache, 
irritability,  insomnia  and  confusion,45  and  it  has  been  shown  further  that 
indol  and  skatol  cause  muscle  to  react  to  stimuli  like  fatigued  muscles.60 
But  the  comparatively  slight  toxicity  can  hardly  be  responsible  for  some 
of  the  symptoms  observed.  Various  explanations  have  been  offered  to 
account  for  the  discrepancy  noted  between  clinical  evidence  of  intestinal 
intoxication  and  the  fact  that  the  substances  formed  thus  far  isolated  are 
only  slightly  toxic.  The  most  plausible  explanation  for  the  discrepancy 
mentioned  is  that  the  list  of  compounds  which  may  be  formed  in  putre- 
faction has  not  yet  been  exhausted,  and  it  is  possible,  and  indeed  probable, 
that  in  time  other  compounds  of  putrefactive  origin  will  be  found  that 
will  adequately  account  for  the  clinical  symptoms  observed.  On  the  other 
hand,  it  be  possible  that  indol  and  skatol  may  exert  quite  different  effects 
on  the  normal  organism  from  those  which  it  exerts  on  a  body  whose  resist- 
ant powers  have  been  lowered  as  a  result  of  other  pathological  processes. 
In  other  words,  the  receptive  condition  of  the  body  under  the  two  condi- 
tions mentioned  may  be  entirely  different,  producing  in  turn  quite  rad- 
ically differing  symptoms. 

Amines  and  Their  Formation.13 — That  we  have  by  no  means  isolated 
all  the  active  principles  from  putrefactive  mixtures  may  be  well  illus- 
trated by  the  investigations  recorded  during  the  last  three  years.  In  1907 
Dixon  and  Taylor29  aroused  considerable  interest  by  the  publication  of 
their  observation  that  alcoholic  extracts  of  the  human  placenta  when 
injected  intravenously  caused  a  marked  rise  in  blood-pressure  and  con- 
tractions of  the  pregnant  uterus.  On  repetition  of  this  work,  Kosenheim87 
failed  to  corroborate  the  findings  of  Dixon  and  Ta}4or  when  extracts  of 
perfectly  fresh  placentas  were  employed.  When,  however,  extracts  of 
placentas  in  various  stages  of  putrefaction  were  intravenously  admin- 
istered, results  were  obtained  identical  with  those  of  Dixon  and  Taylor. 
A  substance  responsible  for  these  effects  has  been  separated  and  identified 
oy  Barger  and  Walpole,15  according  to  whom  the  active  principle  is 
p.  oxyphenylethylamin  and  may  be  derived  from  tyrosin  as  a  result  of  the 
following  reaction:  0H  0H 


CH.  NK 


IcoolH 


13 


Moreover,  iso-amylamin  has  been  isolated  from  putrefactive  mixtures 
probably  being  derived  from  leucin  in  accordance  with  the  following 
reaction : 

CH,      CH-  CH  CH, 

V  V 

CH 


CH.  NH. 
I 

COO  H 


CH 

CHZ 
i 

CH2 
I 


(Leucin)  I s ©any 1 ami n 

These  results  have  led  the  authors  to  remark  that  they  are  induced  "to 
emphasize  the  probability  that  the  amins  which  we  have  isolated  are  nor- 
mally formed  by  putrefaction  in  the  intestine  and  are  absorbed 
from  it."15 

The  compound,  p.  oxyphenylethylamin,  is  of  peculiar  interest  for  sev- 
eral reasons.  In  the  first  place,  it  was  originally  isolated  by  Emerson36 
from  an  autolysis  of  pancreas  and  its  mode  of  formation  from  tyrosin  has 
always  been  considered  unique.  It  is  obvious  at  present  that  it  was  prob- 
ably produced  by  putrefaction  in  this  case  also.  Much  more  interest 
attaches  to  this  substance  from  its  great  resemblance,  both  structurally 
and  pharmacologically,  to  epinephrin. 


OH 

/\ 


OH 


p.oxypAenyierAyJam/',, 


CH.  OH 
( 

CHa 
I 

ad, 


"P.  oxyphenylethylamin  has  an  action  very  similar  to  that  of 
adrenalin  [epinephrin],  reproducing  both  the  motor  and  inhibitory  effects 
of  nerves  of  the  true  sympathetic  system.  It  produces  the  motor  more 
powerfully  than  the  inhibitory  effects.  Its  action  differs  from  that  of 
adrenalin  in  being  weaker,  and  slower  in  onset,  and  in  being  less  strictly 
though  mainly  peripheral.  It  is  absorbed  from  the  subcutaneous  tissues 
and  the  alimentary  canal  and  produces  its  effects  when  so  administered."27 
Isoamylamin  has  a  similar  action. 

Finally,  it  is  of  exceeding  great  interest  to  note  that  p.  oxyphenyl- 
ethylamin is  one  of  the  substances  which  give  to  ergot14  its  characteristic 


14 


action  on  the  uterus.  It  is  also  probable  that  it  is  identical  with  the 
urohypertensin  of  Abelous  and  Bardier.6  These  observations  also  indicate 
the  necessity  for  controlling  all  possible  sources  of  bacterial  contamination 
when  extracts  of  animal  tissues  are  employed  in  demonstration  of  specific 
action.  Again,  "Many  observations  have  been  published  recently  con- 
cerning the  presence  in  the  blood-serum  and  urine  in  various  pathological 
conditions,  of  substances  which  cause  dilatation  of  the  pupil  of  the 
enucleated  eye  of  the  frog.  The  fact  that  both  these  amins  have  this 
action  casts  some  doubt  at  least  on  the  validity  of  the  assumption,  made 
by  certain  observers,  that  the  substance  in  serum,  responsible  for  this 
effect,  is  adrenalin."27  On  introduction  into  the  body,  the  base  is  elimi- 
nated as  p.  oxyphenylacetic  acid,40  another  example  of  deamination. 

OH  OH 


f>.  oty phenyl  ethyl- 


or"' 


Oxy phenyl  • 
acid 


amin 


When  histidin  is  subjected  to  the  action  of  putrefactive  bacteria,  a 
compound7  is  produced  which  holds  promise  of  being  responsible  in  a 
measure  for  certain  reactions  which  have  long  been  unexplained. 

CH 

hn  /"\r 
c 

CH2 
CH^ 

/3  imina^olylethylam'tn 

This  substance,  beta-iminazolylethylamin,  resembles  in  some  respects 
p.  oxyphenylethylamin  in  that  both  compounds  are  contained  in  ergot 
extracts,  and  both  substances  exert  similar  influences  on  the  muscle  of 
the  uterus.  In  addition  to  the  reactions  possessed  in  common  with 
p.  oxyphenylethylamin,  beta-iminazolylethylamin  is  capable  of  calling 
forth  symptoms  practically  identical  with  those  induced  by  injections  of 
peptone  solutions,  or  by  serum  or  other  protein  in  the  sensitized  guinea- 
pig,  that  is,  producing  anaphylactic  shock.27    The  base  has  also  a  mild, 


direct,  stimulant  effect  on  the  activity  of  the  salivary  glands  and  the 
pancreas.  This  secretory  action,  being  paralyzed  by  atropin,  may  be 
regarded  as  a  weak  action  of  the  pilocarpin  type ;  the  association  has  some 
interest  in  that  pilocarpin  also  contains  an  iminazole  ring.  More  recently 
this  base28  has  been  isolated  from  extracts  of  the  intestines,  thus  lending 
support  to  the  suggestion  that  under  normal  conditions  it  may  exert  a 
more  or  less  definite  function  in  the  maintenance  of  nutritional  rhythm. 

Closely  associated  with  p.  oxyphenylethylamin  in  extracts  of  placentas 
is  another  compound  which  may  also  be  considered  as  an  amin  and  which 
has  an  action  antagonistic  to  that  of  p.  oxyphenylethylamin.  This  com- 
pound, cholin,  has  been  the  subject  of  a  great  deal  of  discussion  during 

^■CHa.CH2.0H 

the  last  few  years.  It  undoubtedly  has  its  origin  in  a  lipoid  compound, 
lecithin,  a  constituent  of  practically  all  cells.  It  is,  therefore,  apparent 
that  in  putrefactive  processes  in  the  tissues,  cholin  may  arise  in  relatively 
large  quantity  and,  although  not  highly  toxic  when  given  by  mouth,  it 
may  be  exceedingly  poisonous  when  allowed  to  come  in  contact  with 
nervous  tissue.  Thus,  Donath30  observed  severe  tonic  and  clonic  con- 
vulsions after  cholin  had  been  injected  directly  into  the  cortex  or  under 
the  dura.  This  investigator  offers  the  opinion  that  cholin  may  be  respon- 
sible for  epileptic  convulsions — an  opinion  founded  on  the  fact  that  he 
with  other  investigators43' 86  has  been  able  to  demonstrate  the  presence 
of  cholin  in  large  quantities  in  the  cerebrospinal  fluid  of  epileptics  and  in 
other  conditions  associated  with  destruction  of  nervous  tissue.  In  accord- 
ance with  this  idea,  unsuccessful  attempts95  have  been  made  to  demon- 
strate the  presence  in  the  blood  of  cholin  in  animals  during  the  tetanic 
convulsions  caused  by  complete  removal  of  thyroids  and  parathyroids. 
The  removal  of  the  glands  has  been  shown  to  result  in  marked  changes  in 
certain  areas  of  the  nervous  system,97  and  it  was  assumed  that  these  histo- 
logical changes  were  due  to  chemical  reactions  whereby  cholin  might  be 
liberated  and  produce  a  secondary  effect. 

Chemically  related  to  cholin  is  neurin,  a  substance  easily  formed  from 
cholin  by  oxidation,  which  is  nearly  twenty  times  as  toxic  as  the  latter. 
The  possibility  presents  itself  that  neurin  may  be  formed  from  cholin 
within  the  organism  under  certain  pathological  conditions  and  may  be 
responsible  for  some  of  the  symptoms  characteristic  of  certain  abnormal 
conditions  as  salivation,  vomiting,  diarrhea  and  a  specific  action  in  caus- 
ing arrest  of  respiration.  This  formation  of  neurin  within  the  organism 
and  any  relationship  which  it  may  bear  to  deranged  metabolism  has  never 


16 


been  conclusively  demonstrated.  It  has  been  shown,  however,  that  neurin 
may  be  excreted,  at  times  at  least,  through  the  urine.56 


These  compounds,  together  with  muscarin  and  betain,  constitute  one 
of  the  groups  of  the  ptomains,  so-called,  and  hence  heretofore  have  been 
interesting  chiefly  because  of  their  advent  into  the  organism  through  the 
introduction  of  decomposing  tissue  taken  as  food.  Nevertheless,  inas- 
much as  they  arise  outside  the  body  as  a  result  of  putrefactive  process, 
there  remains  open  the  possibility  that  they  may  be  formed  in  the  alimen- 
tary canal  or  in  tissues  either  in  a  process  of  degeneration  or  putrefaction. 
The  old  idea,  that  there  is  a  hard-and-fast  line  to  be  drawn  between  plant 
alkaloids  and  animal  poisons  is  rapidly  disappearing,  and  the  fact  that 
one  toxic  compound,  as  for  instance  muscarin,  occurs  as  a  rule  in  plant 
tissue  does  not  exclude  the  possibility  of  the  presence  of  the  same  body  as 
a  result  of  chemical  reactions  within  the  animal  organism.  The  best 
recognition  of  this  disappearing  demarcation  line  is  to  be  found  in  the 
new  publication  by  Winterstein  and  Trier,101  where  all  basic  substances, 
whether  of  animal  or  plant  origin,  are  considered  as  alkaloids. 

From  this  review  of  the  more  recently  discovered  compounds  which 
may  arise  within  the  body  by  putrefactive  processes,  one  fact  stands  forth 
with  striking  clearness,  namely,  the  possible  functions  which  some  of  these 
substances  may  exert  in  physiological  processes  and  their  significance  in 
problems  concerned  with  disease.  We  have  seen  that  one  compound  resem- 
bles epinephrin,  both  structurally  and  in  physiological  activity,  while 
another  stimulates  the  activity  called  forth  by  pilocarpin.  Epinephrin  and 
pilocarpin  are  in  daily  use  as  drugs,  the  degree  of  whose  activities  may  be 
regulated  at  will.  The  protein  derivatives  mentioned  have  practically 
similar  actions  but,  as  they  arise  within  the  organism,  can  not  be  subjected 
to  voluntary  control.  It  is  likely  that  under  normal  conditions,  only 
small  quantities  of  these  compounds  may  be  thrown  into  the  blood-stream 
and  that  there  is  some  nice  adjustment  of  mechanism  which  may  have  an 
influence  on  the  further  beneficial  disposition  of  these  bodies.  It  is  con- 
ceivable, however,  that  at  times  an  undue  quantity  of  such  material  may 
overwhelm  the  regulating  mechanism  to  such  a  degree  that  substances 
which  perhaps  normally  aid  in  the  maintenance  of  physiological  rhythm 
may  indeed  become  responsible  for  the  advent  of  abnormal  reactions. 
The  suggestion  is  therefore  offered  that  some  of  the  disturbances  asso- 
ciated with  excessive  intestinal  putrefaction  may  have  such  an  origin.  A 
hypothesis  of  this  sort  readily  furnishes  an  explanation  for  the  vaguely 


cholin 


neurin. 


17 


defined  headache  and  general  malaise  characteristic  of  the  previously  men- 
tioned pathological  states. 

DIAMINES 

Cystinuria  has  been  given  a  great  deal  of  consideration  by  investiga- 
tors for  the  excellent  opportunity  it  afforded  of  elucidating  some  of  the 
complex  processes  underlying  the  principles  of  intermediary  metabolism. 
It  has  been  and  still  is  a  matter  of  great  uncertainty  as  to  the  origin 
and  significance  of  cystin  and  the  diamins,  putrecin  and  cadaverin,  which 
appear  in  the  excretia  of  certain  individuals.  The  appearance  of  cystin 
has  been  the  subject  of  such  widespread  interest,  and  the  results  of  study 
of  cystinuria  have  been  so  extensive  and  are  so  well  known  that  further 
discussion  of  this  subject  in  this  place  appears  superfluous.75 

On  the  other  hand,  the  diamins  have  not  received  so  much  attention. 
The  structure  of  these  bases  is  as  follows : 

CH2 .  CH2 .  CH2 .  CH2  CH2 .  CH2 .  CH2 .  CH2 .  CH2 

NH2  NH2  NH2  NH2 

Tetramethylendiamin  Pentamethylendiamin 
Putrecin  Cadaverin 

A  third  diamin,  neuridin  or  saprin,  is  isomeric  with  cadaverin.  The 
diamins  are  eliminated,  not  only  in  the  urine  but  also  in  the  feces.  As 
has  been  stated  previously,  they  are  found  usually  associated  with  cystin, 
although  the  condition  responsible  for  the  appearance  of  cystin  in  the 
urine  does  not  seem  necessary  for  the  condition  of  diaminuria,  for  these 
substances  have  also  been  eliminated  under  certain  other  pathological 
conditions ;  principally,  intestinal  disturbances,  as  for  instance,  in  various 
infections,  in  cholera,85  dysentery,  gastro-enteritis,84  and  from  one  case  of 
pernicious  anemia  tetramethylendiamin  was  isolated.46  The  origin  of 
these  bodies  appears  to  be  in  two  diamino-acids  that  are  products  of 
normal  digestion. 

From  lysin,  which  has  the  following  formula: 

CH2 .  CH2 .  CH2 .  CH2 .  CH .  COOH 
NH2 

C02  is  split  off,  forming  cadaverin,  with  the  following  formula : 

CH2.CH.2CH2.CH2.CH2 
NHa  NH2 

Putrecin  is  derived  ultimately  from  arginin,  guanidin  and  amino- 
valerianic  acid.  Kossel  and  Dakin55  have  demonstrated  the  existence  of 
an  enzyme,  arginase,  in  certain  tissues  of  the  body  which  is  capable  of 
splitting  arginin  into  urea  and  ornithin.  Thus: 

CH2 .  CH2 .  CH2 .  CH  .  COOH 

NH  NH2  CH2.CH2.CH2.CH.COOH 

I  I  I  * 

C-NH2  NH2  NH2 

|  Ornithin 
NH2 

Arginin 


18 


From  ornithin,  the  formula  for  which  is  as  follows : 

CH2 .  CH2 .  CH2 .  CH .  COOH 
NHa  NH2 

C02  is  split  off,  forming  putrecin,  the  formula  below : 

CH2 .  CH2 .  CH2 .  CH2 
NH2  l!fH2 

In  intestinal  disturbances,  it  is  probable  that  these  compounds  are  the 
result  of  the  bacterial  activity — indeed  they  may  be  the  metabolic  prod- 
ucts eliminated  by  microorganisms.  In  cystinuria,  however,  it  is  possible 
that  a  different63  explanation  for  diaminuria  is  pertinent.  It  may  be 
assumed,  for  instance,  that  in  the  beginning  cystinuria  and  diaminuria 
are  brought  about  through  a  similar,  or  indeed  the  same  cause,  or  causes, 
for  example,  a  gradually  changing  type  of  metabolism  induced  by  some 
unknown  agency,  resulting  in  an  anomaly  of  metabolism.  If  the  anomaly 
is  slight  in  character,  cystin  alone  is  eliminated  as  a  result,  whereas  if  the 
change  in  metabolism  is  sufficiently  pronounced  diamins  are  also  excreted. 
If  this  assumption  is  accepted,  it  is  easy  to  explain  why  in  some  cases  of 
cystinuria  the  diamins  are  absent,  and  that  gradually  one  or  both  of  these 
compounds  disappear,  that  cystinuria  persists,  but  that  cystinuria  does 
not  cease  and  leave  diaminuria.  Thus  far  all  attempts  to  produce 
diaminuria  experimentally  have  been  unsuccessful.  These  bodies  are 
possessed  of  a  certain  interest,  aside  from  their  chemical  significance  in 
that,  like  nearly  all  the  amins,  they  are  more  or  less  toxic.  It  has  been 
demonstrated  experimentally  that  the  diamins  may  also  exert  an  influ- 
ence on  certain  intermediary  processes.  Thus,  according  to  Pohl,81  feed- 
ing diamins  inhibits  certain  well-known  protective  reactions  which  the 
organism  is  capable  of  putting  forth,  as  for  instance,  the  formation  of 
glycuronates  and  the  synthesis  of  hippuric  acid. 

The  toxicity  of  these  compounds  calls  to  mind  the  poisonous  action 

NH2 

of  another  diamin,  not  found  in  the  organism,  namely,  hydrazin,94  ^ 

This  substance  is,  however,  very  much  more  toxic  than  the  diamins  just 
mentioned.  Its  introduction  into  the  organism  is  followed  by  marked 
histological  changes"  in  various  tissues,  especially  in  the  liver.  In  fact, 
the  action  of  this  substance  is  directed  almost  specifically  on  the  liver, 
provoking  fatty  degeneration  of  that  organ.  Only  the  cytoplasm  of  the 
cell  is  attacked.  Although  the  liver  is  almost  completely  transformed 
under  the  influence  of  hydrazin,  no  noticeable  change  in  nitrogenous 
intermediary  metabolism  can  be  demonstrated  through  a  study  of  the 
urinary  constituents. 


11) 


APORRHEGMAS 

In  a  recent  communication  Ackermann  and  Kutscher8  have  proposed 
a  new  designation  for  the  transformation  products  of  amino-acids  which 
are  formed  by  life  processes,  whether  in  the  animal  or  the  vegetable 
kingdom.  The  term  employed  for  these  bodies  is  "aporrhegma."  It  is 
interesting  to  note  the  number  of  such  compounds  which  may  arise  from 
putrefaction  alone.  The  above-mentioned  authors8'  9  have  given  a  list  of 
these  substances,  together  with  the  amino-acids  from  which  they  are 
derived. 

AMINO-ACID  APORRHEGMA 

Histidin   Iminazolylethylamin 

Iminazolpropionic  acid 

Arginin   Ornithin 

Tetramethylendiamin 
Aminovalerianic  acid 

Lysin   Pentamethylendiamin 

Glutaminic  acid   Aminobutyric  acid 

Asparaginic  acid  Alanin 

Succinic  acid 

Glycocoll   Methylamin   (  ? ) 

Leucin   Isoamylamin 

Isovalerianic  acid 

Prolin   Pyrrolidin 

Phenylalanin   Phenylethylamin 

Phenylacetic  acid 
Phenylpropionic  acid 

Tyrosin   Oxyphenylethylamin 

Oxyphenylacetic  acid 
Oxyphenylpropionic  acid 

Tryptophan   Indol 

Skatol 

Indolpropionic  acid 
Indolacetic  acid 


METHYLATION  IN  THE  ORGANISM 

Until  the  last  year  or  two,  methylation  within  the  organism  was 
looked  on  as  a  reaction  which  occurred  only  rarely.  With  the  exception 
of  the  well-known  examples  of  the  methylation  of  tellurium66  and 
selenium,  our  knowledge  of  this  type  of  physiological  activity  was  exceed- 
ingly limited.  Renewed  interest  in  the  process  of  methylation  has  been 
aroused  by  the  recent  investigations  of  Engeland,37  who  has  demonstrated 
that  complete  methylation  of  most  of  the  amino-acids  derived  from  the 
protein  molecule  is  far  from  a  difficult  reaction.  The  betains  comprise 
all  that  group  of  bodies  of  the  aliphatic  series  which  are  basic  in  character, 
thus,  such  compounds  as  the  various  amins,  cholin,  muscarin,  urea, 
creatin,  etc.  It  is  further  probable  that  the  wide-spread  distribution  of 
the  betains,  both  in  the  animal  and  vegetable  kingdoms,  is  to  be  explained 
solely  by  decomposition  of  protein.  Moreover,  it  has  been  suggested  that 
all  the  betains  which  arise  from  amino-acids  formed  by  decomposition  of 
protein  bear  a  relation  to  the  so-called  "alkaloids." 


20 


A  methylated  amino-acid  was  unknown  in  the  animal  kingdom  until 
completely  methylated  glycocoll  was  isolated  in  considerable  quantity 
from  crab  meat.8  Accompanying  methyl  glycocoll  was  found  trimethyl- 
amin  oxid.91 

CHa. COOH  J 
Methyl  glycocoll  Trimethylamin  oxid 

Under  normal  conditions  methylation  of  amino-acids  does  not  occur 
to  any  considerable  extent  in  the  body  of  the  warm-blooded  animals, 
owing  probably  to  the  fact  that  if  such  compounds  are  formed,  they  are 
at  once  oxidized  and  serve  as  energy-producing  material.  When  abnormal 
conditions  are  induced,  however,  as  in  phosphorus  poisoning,  where, 
perhaps,  oxidative  processes  are  less  active  the  appearance  of  methylated 
compounds  may  be  observed.  Thus,  the  methylated  gamma  amino- 
butyric  acid  has  been  isolated  recently  from  the  urine  of  a  dog  poisoned 
with  phosphorus.38  This  substance  probably  arises  from  glutaminic  acid, 
being  transformed  into  gamma  amino-butyric  acid,  then  completely 
methylated  and  eliminated. 


coo 
*— - 

CH.NH,  CH, 
/         2  «  ' 

CHa   >►  CH, 


CHa.NH2 
I 


CHj,  COOH 

COOH  J  amino-butyric     methylated  y  ami no- 

Glutafliinic  acid  acid  butyric  acid 

In  plants  and  the  lower  animals  methylated  glycocoll  is  an  amino-acid 
which  is  very  widely  distributed.  Glycocoll  is  exceedingly  resistant  to 
decomposition  by  putrefaction,  as  was  long  ago  demonstrated  by  Nencki. 
It  is  the  only  amino-acid  which  appears  regularly  in  the  urine,  in  the 
form  of  hippuric  acid.  It  is  found  in  its  mono-methylated  form  as 
sarcosin  combined  with  the  guanidin  residue,  or  modified  urea  rest, 
forming  creatin  of  the  muscle  or  creatinin  of  the  urine  of  the  higher 
animals. 

NH2  NH  CO 

NH2  NH.CH3  I  I  I 

|  |  C-NH  C-NH 

CH2 


C-NH  C-NH 
CH2                           |                         I  I 
|  H3C-N  H3C-N  CH2 


COOH  Creatinin 
Glycocoll  Methyl  CH2 

glycocoll 

COOH 
Creatin 

Creatin  and  Creatinin.68' 72 — At  present,  perhaps  the  most  interesting 
example  of  methylation  may  be  found  in  the  origin  of  the  creatin  of 
muscle  and  the  creatinin  of  the  urine.    The  exact  significance  of  these 


21 


compounds  is  exceedingly  obscure.  It  is  known,  for  example,  that  under 
physiological  conditions  creatin  is  absent  from  the  urine  and  that  creatinin 
elimination  is  practically  constant  for  a  given  individual.  This  elimina- 
tion, however,  is  different  for  different  individuals,  but  bears  no  relation 
to  the  volume  of  the  urine  or  the  total  nitrogen  excreted.  From  a  long 
series  of  investigations,  it  has  been  concluded  that  creatinin  is  an  index 
to  some  special  form  of  normal  metabolism,  as  yet  unknown,  but 
undoubtedly  connected  with  processes  concerned  with  muscle  tissue. 
There  is  apparently  a  somewhat  close  relationship  between  muscle 
efficiency  and  creatinin  elimination.  Creatinin  excretion  varies  greatly 
under  abnormal  conditions,  being  increased  in  fevers  and  diminished  in 
a  large  number  of  other  pathological  states.  Particularly  striking  is  the 
diminution  in  excretion  in  abnormal  metabolism  of  muscle  tissue  and  of 
the  liver. 

Ordinarily,  creatin  is  absent  from  the  urine,  but  may  be  present  in 
large  quantities  under  certain  pathological  conditions,  especially  those 
associated  with  inanition,  depression  of  liver  function,  or  an  abnormal 
state  of  muscle.  The  appearance  of  creatin  in  the  urine  under  these 
circumstances  might  lead  one  to  infer,  by  analogy  with  the  presence  of 
the  betain  of  amino-butyric  acid  noted  previously,  that  when  abnormal 
processes  are  in  order,  certain  chemical  reactions  are  held  in  abeyance 
resulting  in  the  elimination  of  creatin  as  an  incompletely  disintegrated 
intermediary  product.  From  the  most  recent  investigations22  concerning 
creatin  and  creatinin,  it  appears  likely  that  there  is  an  intimate  relation- 
ship between  these  substances  and  carbohydrate  metabolism,  although, 
at  present,  this  relationship  is  not  more  than  a  mere  indication.  For 
example,  creatin  is  present  in  the  urine  during  fasting  both  in  man  and 
animals.  Administration  of  carbohydrate  under  these  circumstances 
causes  the  rapid  disappearance  of  urinary  creatin.  It  has  also  been 
suggested  that  one  function  of  creatin  being  a  base,  is  to  serve  as  an 
alkali  in  the  muscle  to  neutralize  lactic  acid  which  arises  as  a  result  of 
muscle  activity.18  Future  investigations  will,  undoubtedly,  reveal  the 
significance  of  the  interrelation  of  creatin,  creatinin,  muscle  and  carbo- 
hydrate and  if  the  prophecy  of  Folin,  uttered  a  few  years  ago  before  the 
Harvey  society,  comes  true,  the  unraveling  of  this  mystery  will  mean 
much  in  the  domain  of  pathology. 

THE  PROTEOSES 

Since  the  classic  experiments  of  Schmidt-Muhlheim,89  the  behavior 
of  the  proteoses  when  injected  into  the  blood  stream  has  occupied  the 
attention  of  a  long  series  of  investigators,  the  aim  of  whom  has  been  to 
explain  the  significance  of  the  reactions  induced.  It  had  been  assumed 
for  years  previous  to  recent  times  that  when  proteoses  were  formed  in 
digestion,  they  were  absorbed  into  the  portal  vein,  carried  to  the  liver 
and  there  were  detoxicated.    As  a  matter  of  fact,  it  was  also  discovered 


22 


that  proteoses  introduced  into  the  portal  vein  were  incapable  of  provoking 
the  typical  effects  induced  by  injection  into  the  systemic  circulation,  an 
observation  in  entire  accord  with  the  then  current  views  regarding  the 
processes  of  digestion  and  absorption. 

Eenewed  activity  directed  toward  the  solution  of  the  problem  con- 
cerning the  normal  presence  of  proteoses  in  the  blood  was  awakened  by 
the  more  recent  investigations  having  to  do  with  the  degradation  of  the 
protein  molecule  in  the  enteric  tract.  According  to  one  school,  proteoses 
are  absorbed  into  the  blood,  thus  casting  doubt  on  the  prevalent  idea  that 
proteoses  are  broken  into  their  simplest  cleavage  products,  which  are 
absorbed,  and  then  further  worked  over  into  the  needful  compounds. 
Opposed  to  this  theory  is  the  school  that  is  unable  to  find  any  evidence  of 
proteoses  in  the  blood  normally.  Amid  these  conflicting  views  and 
observations,  the  apparent  consensus  of  opinion  is  that  proteoses  are 
absent  from  the  blood  under  normal  conditions.19  Hence,  when  these 
substances  are  introduced  into  the  blood-stream,  they  act  as  poisons 
calling  forth  certain  characteristic  reactions,  fever,  fall  of  pressure, 
changes  in  respiration,  increased  flow  of  lymph,  saliva,  and  other  secre- 
tions, but  causing  a  somewhat  prolonged  anuria.  There  has  been  con- 
siderable controversy  as  to  whether  pure  proteoses  are  really  toxic.  All 
the  older  observations  were  subjected  to  severe  criticisms  by  Pick  and 
Spiro,80  who  maintained  that  proteoses  as  such  are  non-toxic  and  that 
the  toxic  principle  is  merely  an  adhering  contamination,  derived  from 
the  animal  enzymes  employed  in  the  preparation  of  the  proteoses. 
According  to  these  authors,  this  substance,  peptozyme,  can  be  rendered 
non-toxic  by  subjecting  the  proteoses  to  a  certain  chemical  treatment. 
Later  observations,96  however,  have  demonstrated  that  this  conclusion 
is  erroneous,  since  proteoses  prepared  from  the  action  of  vegetable 
enzymes  on  vegetable  proteins  and  also  naturally  occurring  vegetable 
proteoses  induce  the  same  train  of  symptoms  as  proteoses  made  from 
animal  proteins  and  enzymes.  Moreover,  cleavage  of  proteoses  beyond  the 
biuret-yielding  stage  does  not  produce  any  symptoms.  From  the  obser- 
vations of  Popielski,  it  is  concluded  that  the  substance  responsible  for 
the  so-called  proteose  action  may  be  removed  in  large  measure  by  treat- 
ment with  alcohol.  This  compound  has  been  designated  vasodilatin  and, 
according  to  Popielski,82  is  not  protein  in  nature.  The  fact,  however, 
that  this  investigator  has  not  succeeded  in  separating  the  vasodilatin 
from  proteoses,  together  with  the  well-known  fact  that  a  portion  of  the 
proteoses  are  soluble  in  alcohol,  militate  against  the  correctness  of  the 
view  that  the  proteoses  action  is  separable  from  these  bodies.  On  the  other 
hand,  it  has  been  suggested28  that  a  portion  of  the  reactions  provoked 
by  "peptone"  may  be  induced  by  the  presence  in  the  "peptone"  of  beta 
iminazolylethylamin  which  is  capable  of  calling  forth  symptoms  in  nearly 
every  respect  similar  to  those  produced  by  the  "peptone."    The  single 


23 


exception  noted  is  that  beta  iminazolylethylamin  does  not  render  the 
blood  non-coagulable,  which  is  distinctly  characteristic  for  "peptone." 

In  our  laboratory  the  observation  has  been  made  recently  that  the 
intravenous  injection  of  proteoses  produces  a  significant  glycosuria. 
Coincident  with  the  appearance  of  sugar  in  the  urine,  there  is  a  marked 
hyperglycemia.  The  cause  and  possible  significance  of  this  reaction  is 
being  investigated.  It  appears  a  little  strange  that,  of  the  long  series  of 
investigations  carried  out  with  the  proteose  injections,  in  not  a  single 
instance  is  there  a  recorded  observation  indicating  glycosuria.  When 
proteoses  are  injected  into  the  blood-stream,  the  major  portion  of  these 
compounds  promptly  reappears  in  the  urine,  although  there  is  some 
evidence  that  they  may  be  partially  transformed  into  smaller  molecules.23 
Albumosuria,  so-called,  is  indicative  of  the  formation  of  proteoses  within 
the  organism  and  is  considered  of  importance  in  diagnosis.  Thus, 
albumosuria  may  be  observed  in  suppuration  of  all  kinds,  resolution  of 
pneumonia,  involution  of  the  uterus,  carcinoma,  atrophy,  eclampsia, 
leukemia,  absorption  of  simple  and  inflammatory  exudates,  febrile 
conditions  with  destruction  of  tissue,  and  ulcerating  pulmonary 
tuberculosis.100 

It  is  possible,  and  indeed  may  be  probable,  that  the  proteoses  formed 
within  the  body  and  thrown  into  the  blood-stream  may  be  responsible  for 
some  of  the  symptoms  which  are  characteristic  of  some  of  the  abnormal 
conditions  cited.  The  fact  that  these  substances  are  fairly  toxic  is  almost 
evidence  for  the  possibility  just  indicated.  The  significance  of  these 
compounds,  both  in  physiology  and  pathology,  warrants  further  investi- 
gation of  the  cause  of  the  symptoms  induced.  It  is  hardly  probable  that 
any  one  specific  group  in  these  complex  polypeptids  is  responsible  for  all 
the  reactions  noted  and  future  study  will  undoubtedly  demonstrate  the 
presence  of  several  distinct  entities  which  specifically  call  forth  certain 
symptoms. 

The  modern  conception  of  the  living  cell  makes  enzymes  responsible 
for  the  numerous  types  of  known  activities;  thus  we  speak  of  enzymes 
facilitating  reduction,  oxidation,  deamination,  cleavage,  etc.  One  organ 
is  found  to  contain  enzymes  active  to  a  high  degree  in  one  direction, 
another  organ  in  another  direction,  and  still  a  third  organ  which  may 
furnish  an  almost  unlimited  number  of  enzymes  capable  of  performing 
varied  types  of  reactions.  Ordinarily  when  enzymes  are  mentioned,  one 
thinks  involuntarily  of  the  best-known  agents,  the  digestive  ferments. 
Our  ideas  concerning  the  exact  mode  of  action  of  the  enzymes  that 
undoubtedly  play  an  important  role  in  intermediary  metabolism  are  not 
well  defined.  It  is  probably,  however,  not  an  unwarranted  position  to 
assume  that  the  several  types  of  reactions  discussed  in  previous  portions 
of  this  paper  have  enzyme  activity  as  their  basis. 

With  the  acceptance  of  enzyme  activity  as  the  foundation  of  cellular 
chemical  activity,  hence,  intermediary  processes,  it  is  easy  to  conceive 


24 


how  the  induction  of  abnormal  environment  for  these  agents  may  lead  to 
the  production  of  distinctly  pathological  phases  of  metabolism  with 
consequent  injury  to  the  whole  physiological  economy.  In  our  endeavor 
to  discover  the  reason,  or  the  cause,  for  abnormal  metabolism  we  have 
been  led  into  the  error  of  looking  for  changes  of  too  great  magnitude. 
Enzymes  are  exceedingly  sensitive  to  all  sorts  of  changes  in  environmental 
conditions.  Too  much  acid,  or  too  little,  lack  of  the  requisite  inorganic 
salts,  perhaps  absence  of  carbohydrate — that  group  of  substances  vaguely 
designated  as  co-enzymes — may  retard  or  hold  in  abeyance  certain  types 
of  reaction  the  effects  of  which,  over  a  considerable  period  of  time,  work 
injury  to  other  types  of  enzymes  until  finally  a  large  number  of  processes 
may  be  carried  out  only  imperfectly.105  Perhaps  one  of  the  best  examples 
of  the  point  under  discussion  may  be  taken  from  the  recent  work  of 
By  waters,20  who  found  that  the  inverting  power  of  an  aqueous  extract  of 
yeast  is  increased  ten-  to  fifteen-fold  by  the  addition  of  acetic  acid.  On 
the  other  hand,  this  activity  given  by  acid  addition  is  susceptible  of 
removal  by  alkalies,  which  subsequent  addition  of  acid  is  capable  of 
counteracting.  Under  one  form  of  environment  the  power  disappears 
and  when  changed  to  another  the  power  is  restored. 

With  data  of  this  type  at  hand,  is  one  unwarranted  in  suggesting  the 
probability  that  pathological  metabolism  of  various  types  may  be  the 
direct  result  of  changed  environmental  conditions  of  intracellular 
enzymes  ?  I  believe  that  such  a  viewpoint  will  do  much  toward  a  solution 
of  many  problems  concerning  metabolism  both  from  the  standpoint  of 
physiology  and  pathology,  the  dividing  line  of  which  is  exceedingly 
narrow. 

The  advances  of  the  future  are  to  be  made,  I  believe,  by  a  careful 
study  of  small  changes,  details  which  at  first  thought  perhaps  appear 
insignificant  but  if  followed  will  lead  to  far-reaching  results.  One  of  the 
best  examples  that  can  be  cited  in  this  connection  may  be  taken  from  a 
recent  paper  on  "The  Influence  of  Alcohol  on  Nitrogenous  Metabolism" 
by  Mendel  and  Hilditch.69  As  a  result  of  their  study,  the  authors 
conclude  that  "the  most  significant  impression,  perhaps,  which  the 
analytical  data  afford,  is  the  absence  of  pronounced  alterations  indicative 
of  markedly  disturbed  protein  metabolism."  Emphasis  on  the  words 
"absence  of  'pronounced  alteration"  shows  an  entire  appreciation  of  the 
presence  of  small  differences.  In  this  particular  case,  the  organism  was 
capable  of  using  certain  doses  of  alcohol  to  its  distinct  advantage; 
beyond  this  limit  changes  in  purin  output  were  observed.  The  cellular 
mechanism  was  altered  in  such  a  manner  that  certain  types  of  processes 
were  changed  in  a  measure,  the  extent  of  the  change  being  indicated  by 
the  altered  purin  output.  The  changed  output  was  not  large,  but  was  it 
not  just  as  good  an  indication  of  deranged  metabolism  as  if  the  change 
had  been  twice  as  great?  If  the  view  suggested  is  correct,  then  the  hope 
of  future  advances  along  the  line  of  intermediary  metabolism  lies  in  an 


25 


appreciation  of  the  significance  of  small  differences  and  changes  in  the 
environmental  conditions  of  the  cell. 
445  Orange  Street. 

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26 


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65.  Malfatti:  Ztschr.  f.  physiol.  Chem.,  1909,  lxi,  499. 

66.  Mead  and  Gies:  Am.  Jour.  Physiol.,  1901,  v,  105. 

67.  Meltzer:  Jour.  Am.  Med.  Assn.,  1907,  xlviii,  655. 

68.  Mendel:   Science,  1909,  xxix,  584. 

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72.  Myers:  Am.  Jour.  Med.  Sc.,  Feb.,  1910. 

73.  Myers  and  Fischer:  Zentralbl.  f.  d.  ges.  Physiol.  u.  Path.  d.  Stoffwechs., 
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[Reprinted  from  Science,  N,&,  Vol.  XXXIV.,  No. 
882,  Pages  722-732,  November  24,  1911] 


THE  ROLE  OF  DIFFERENT  PROTEINS  IN  NUTRITION 
AND  GROWTH 

Interest  in  the  study  of  the  problems  of 
nutrition  has  largely  been  coincident  with  the 
development  of  the  chemical  aspects  of  physi- 
ology, in  distinction  from  the  physical  and 
mechanical  phenomena  which  earlier  attracted 
the  attention  of  investigators.  The  subject 
of  nutrition  has,  in  large  measure,  been  con- 
sidered in  the  past  from  what  might  be  desig- 
nated as  a  statistical  standpoint.  The  balance 
of  income  and  outgo  of  energy  and  matter, 
nutritive  needs  and  dietary  standards,  and  the 
effect  of  external  factors  on  these,  are  illustra- 
tions of  the  type  of  questions  which  has  called 
for  discussion.  With  the  progress  in  the  study 
of  physiological  chemistry  have  come  impor- 
tant additions  to  our  knowledge  of  the  make- 
up of  the  foodstuffs  and  of  the  real  signifi- 
cance of  the  processes  which  take  place  in  the 
alimentary  tract.  The  conception  of  digestion 
as  a  simple  act  of  solution  has  evolved  into 
that  of  an  intricate  and  carefully  regulated 
chemical  transformation.  The  intermediary 
changes  which  characterize  the  metabolism  of 
food  materials  after  absorption  and  incident 
to  the  real  nutritive  reactions  of  the  body 
within  its  tissue  cells  have  at  length  become 
the  subject  of  experimental  inquiry. 

With  this  development  has  come  about  an 
appreciation  of  the  specific  role  of  foodstuffs. 
Various  incidents  have  favored  this  trend  of 
physiology.  The  study  of  enzymes  and  their 
striking  specificity  has  served  to  emphasize  the 
necessity  of  digestion  before  the  nutrients  can 
satisfy  their  purposes.    Observations  on  the 


2 


unique  responses  of  various  parts  of  the  ali- 
mentary tract  to  different  kinds  of  chemical 
compounds  have  brought  to  light  the  remark- 
able interrelations  of  the  secretory  and  motoi 
functions  of  the  digestive  tract  and  their  de- 
pendence on  special  (chemical)  stimulants. 
But  more  important  than  all  this,  perhaps, 
have  been  the  disclosures  of  the  past  decade  in 
respect  to  the  chemical  structure  of  the  so- 
called  proximate  principles,  and  the  proteins 
in  particular.  The  development  of  this  field 
of  study  has  been  little  short  of  epoch  making, 
so  that  it  seems  timely  to  begin  to  apply  some 
of  the  newer  knowledge  to  the  investigation  of 
problems  in  nutrition. 

The  idea  that  proteins  of  different  origin 
may  possess  an  unlike  physiological  value  is 
not  entirely  new.  Gelatin,  for  example,  has 
long  been  pointed  out  as  an  illustration  of  an 
inadequate  protein.  It  has  been  impossible 
experimentally  to  sustain  life  with  a  diet  in 
which  gelatin  formed  the  sole  source  of  nitro- 
genous intake.  To-day  one  can  cite  other 
illustrations  of  proteins,  e.  g.,  zein,  gliadin, 
hordein,  casein,  which  lack  some  of  the  char- 
acteristic amino-acid  complexes  readily  ob- 
tainable from  other  albuminous  materials 
which  are  vaguely  regarded  as  "  complete." 
In  still  other  cases,  e.  g.,  edestin  and  glutenin, 
the  relative  proportions  of  these  constituent 
complexes  are  so  markedly  different  from  the 
average  as  to  raise  the  question  of  compara- 
tive nutrient  values.  Overabundance  of  glut- 
aminic  acid  groups  must  necessarily  be  at- 
tended by  relative  deficiency  in  other  so-called 
"  building  stones  "  of  the  protein  fundament. 
If,  then,  a  minimum  of  some  of  these  is  an 
indispensable  requirement  of  tissue  mainte- 
nance or  growth  or  repair,  problems  of  relative 
values  at  once  suggest  themselves.1    To  this 

1  These  and  related  questions  are  discussed  in 
detail  by  Mendel,  "Ergebnisse  der  Physiologie, ' ' 
1912,  XL 


8 


may  be  added  the  question  of  protein  synthesis 
in  animals  which  has  been  so  vigorously  de- 
bated in  recent  years.  Here  we  touch  upon 
problems  quite  independent  of  the  energy 
needs  of  the  organism,  yet  equally  important. 
No  sooner  has  the  idea  of  the  isodynamic  re- 
placement of  nutrients  found  acceptance,  than 
the  practical  limitations  of  this  law  are  sub- 
jected to  critical  examination. 

The  foremost  reason  why  so  little  is  known 
in  the  directions  noted  lies  in  the  fact  that  the 
individual  foodstuffs  have,  with  very  few  ex- 
ceptions, rarely  been  examined  heretofore  in 
respect  to  their  actual  nutrient  role.  Meat 
and  cereals  have,  it  is  true,  been  crudely  an- 
alyzed in  terms  of  protein  (N  X  6.25),  fat, 
carbohydrate  and  ash,  and  fed  as  assumed 
mixtures  of  the  composition  indicated.  Physi- 
ologists are,  however,  just  beginning  to  recog- 
nize the  extreme  chemical  complexity  of  such 
animal  and  plant  tissues.  How  much  of  the 
nutritive  failures  or  successes  shall  be  ascribed 
to  either  presence  or  paucity  of  some  inci- 
dental component,  as  lime  or  iron,  as  lipoid  or 
nitrogenous  "  extractive "  of  specific  physi- 
ological import,  such  as  is  attributed  to  the 
"  hormones  "  % 

It  is,  indeed,  only  in  very  recent  years  that 
the  perfection  of  biochemical  technique  has 
permitted  the  preparation  of  isolated  proteins 
in  what  may  be  called  comparative  purity. 
We  believe,  from  the  experience  which  one  of 
us  has  gained  during  many  years  of  experi- 
ment in  this  field,  that  the  vegetable  proteins 
to-day  are  in  general  easier  of  access  for 
chemical  investigation  and  isolation  than  the 
related  compounds  of  animal  origin.  And  it 
is  this  fact  which  encouraged  us  to  undertake 
what  Carl  Voit  long  ago  proclaimed  as  the 
ideal  method,  viz.,  the  feeding  of  isolated  food- 
stuffs under  controllable  conditions.  The  la- 
borious and  costly  investigations  which  are 
under  way  have  been  made  possible  by  the 


4 


cooperation  of  the  Carnegie  Institution  of 
Washington.  A  detailed  report  of  the  first 
two  years'  work  and  the  literature  pertaining 
thereto  is  available  in  Publication  156,  Parts 
I.  and  II.,  of  the  Carnegie  Institution.2  The 
following  pages  are  intended  to  call  attention 
very  briefly  to  some  aspects  of  these  studies. 

We  have  undertaken  to  investigate  certain 
features  of  nutrition  by  feeding  isolated  food 
substances  to  albino  rats.  The  selection  of 
this  animal  has  been  determined  by  several  in 
part  obvious  considerations.  The  white  rat  is 
easily  reared  and  cared  for.  Its  small  size 
reduces  the  food  requirement  to  a  magnitude 
which  falls  within  the  range  of  experimental 
possibility  where  the  preparation  of  special 
dietaries  by  laborious  processes  is  a  funda- 
mental prerequisite.  Furthermore,  the  longev- 
ity of  this  animal  is,  according  to  Donaldson, 
about  three  years ;  so  that  the  first  year  of  life 
corresponds  to  a  long  span  in  terms  of  human 
years.  Not  insignificant  is  the  additional  fact 
that  the  white  rat  has  in  recent  years  been 
made  the  subject  of  exceptionally  extensive 
measurements  in  respect  to  growth  and  vari- 
ous features  of  development  at  the  Wistar 
Institute  in  Philadelphia.  In  this  way  phys- 
ical standards,  so  to  speak,  have  been  estab- 
lished for  this  animal. 

At  the  outset  numerous  problems  of  experi- 
mentation have  arisen  quite  apart  from  the 
main  question  itself.  Can  rats  be  kept  in 
health  indefinitely  under  cage  conditions  which 
permit  the  control  of  the  food  intake  and  col- 
lection of  the  excreta  ?  For  the  description  of 
the  cages  and  experimental  technique  we  must 
refer  to  our  detailed  publication  (Part  I.). 
How  successful  this  has  been  is  best  answered 

2  "Feeding  Experiments  with  Isolated  Food- 
substances,"  by  Thomas  B.  Osborne  and  Lafay- 
ette B.  Mendel,  with  the  cooperation  of  Edna  L. 
Ferry,  Carnegie  Institution  of  Washington,  Publi- 
cation 156,  Parts  I.  and  II.,  1911. 


5 


by  the  statement  that  rats  have  been  main- 
tained for  many  months  at  all  ages  with  ap- 
parent success.  Far  more  important  than  the 
ability  to  withstand  confinement  in  a  re- 
stricted space  has  been  the  demonstration  of 
the  possibility  of  maintaining  rats  on  an 
"  artificial "  food  paste  of  unaltered  uniform 
composition  during  a  large  span  of  their  life. 
Herein  we  have  apparently  been  far  more  suc- 
cessful than  any  of  our  predecessors;  for  the 
supposed  monotony  of  diet  has  been  the 
stumbling  block  leading  to  failure  in  the 
records  of  various  investigators.3  Their  ani- 
mals have  failed  to  eat  and  have  declined  as 
an  obvious  result  of  insufficient  food  intake. 
We  are  inclined  to  lend  emphasis  to  the  result 
of  the  excellent  hygienic  environment  and  care 
of  our  animals.  And  whereas  nutritive  de- 
cline has  commonly  been  attributed  to  the 
anorexia  consequent  upon  the  monotony  of 
diet,  we  are  more  than  ever  inclined  to  shift 
the  explanation  in  many  such  cases  to  mal- 
nutrition as  a  primary  cause.  From  this  point 
of  view  improper  diet  and  malnutrition  may 
be  the  occasion  rather  than  the  outcome  of  the 
failure  to  eat — a  distinction  perhaps  not  suffi- 
ciently recognized  heretofore. 

As  the  criterion  of  the  nutritive  status  of 
the  rats  their  body  weight  has  been  adopted, 
and  this  has  proved  to  be  an  advantageous 
index.  It  soon  became  apparent  that  one  must 
distinguish  sharply  between  maintenance  and 
growth  in  any  such  study  of  nutrition.  The 
white  rat  shows  a  very  characteristic  curve  of 
growth  (plotted  from  the  body  weight)  which 
becomes  practically  stationary  within  300 
days.  According  to  Donaldson  the  body  weight 
changes  from  5  grams  at  birth  to  270  grams 
in  the  case  of  the  male,  or  225  grams  in  the 
female  at  the  age  of  300  days.    To  judge  of 

8  These  earlier  studies  are  reviewed  in  Publica- 
tion 156,  Part  I.,  Carnegie  Institution  of  Wash- 
ington, 1911. 


6 


the  effect  of  a  dietary  regime  by  noting  the 
subsequent  duration  of  life,  as  is  still  fre- 
quently done  by  investigators,  is  misleading; 
for  the  incidence  of  death  may  depend  on  the 
previous  nutritive  condition — the  store  of  fat 
and  glycogen — where  food  is  insufficient.  An 
error  less  readily  appreciated  consists  in  de- 
scribing the  nutritive  status  as  necessarily 
satisfactory  because  an  animal  maintains  an 
undiminished  body  weight  over  long  periods 
under  the  conditions  imposed.  A  man  who 
maintains  his  weight  may  be  in  excellent  nu- 
tritive condition ;  but  a  child  which  does  like- 
wise is  failing  to  grow.  Childhood  demands 
of  a  perfect  ration  the  possibility  of  normal 
growth,  not  simply  maintenance.  This  can 
not  be  emphasized  too  strongly.  Furthermore, 
growth  in  the  sense  of  an  increase  in  the  size 
of  some  structural  part  of  the  body  or  some 
organ  may  proceed  independently  of  the  cor- 
related development  of  the  body  as  a  whole. 
Even  with  the  existence  of  unquestionable 
malnutrition,  skeletal  growth  may  manifest 
itself  in  a  conspicuous  degree;  so  that  the 
length  or  height  of  an  individual  may  mark- 
edly increase  while  the  total  body  weight  re- 
mains stationary  or  even  declines.  One  part 
of  an  organism  may  thrive  at  the  expense  of 
other  tissues.  The  complexity  of  these  rela- 
tionships of  absolute  and  relative  (or  propor- 
tionate) growth  have  likewise  commanded  at- 
tention in  our  experiments. 

A  study  of  physiological  literature  will 
make  it  evident  that  no  convincing  reply  has 
been  given  to  the  question:  can  life  be  main- 
tained and  is  growth  possible  with  a  single 
protein  in  the  dietary.  "Protein"  has  been 
used  in  this  connection  in  a  generic  sense; 
and  one  of  the  (chemically)  simplest  foods, 
milk,  contains  at  least  two  proteins  of  marked 
individuality.  Casein  and  lactalbumin  are 
chemically  unlike.    How  widely  two  exten- 


7 


sively  used  food  proteins  may  differ  in  their 

chemical  make-up  is  indicated  below. 

The  individuality  of  proteins  of  different 

biological  origin  is  further  indicated  by  their 

Casein4  Ze:iir- 
Ammo-acids  Per  Cent.         Per  Cent. 

Glycocoll    0.00  0.00 

Alanine    1.504  9.79 

Valine    7.204  1.88 

Leucine    9.356  1  9.55 

Proline    6.707  9.04 

Asparticacid    1.394  1.71 

Glutaminic  acid    15.554  26.17 

Phenylalanine    3.208  6.55 

Tyrosine    4.509  3.55 

Serine    0.5010  1.02 

Oxyproline    0.2310   

Histidine    2.50u  0.82 

Arginine    3.81u  1.55 

Lysine    5.95u  0.00 

Tryptophane    1.508  0.00 

Diaminotrioxydodecanic  acid  0.7512   

Ammonia    1.61^  3.64 

Sulphur    0.7614  0.60 

Phosphorus    0.8514  0.00 

4  Osborne  and  Guest,  Journal  of  Biological 
Chemistry,  1911,  IX.,  p.  333. 

'Osborne  and  Liddle,  American  Journal  of 
Physiology,  1910,  XXVI.,  p.  304. 

'Levene  and  Van  Slyke,  Journal  of  Biological 
Chemistry,  1909,  VI.,  p.  419. 

7  Van  Slyke,  Berichte  der  deutschen  chemischen 
Gesellschaft,  1910,  XLIV.,  p.  3170. 

8  Abderhalden,  Zeitschrift  fur  physiologische 
Chemie,  1905,  XLIV.,  p.  23. 

•Beach,  Virchow's  Archiv,  1899,  CLVIIL,  p.  288. 

10  Fischer,  Zeitschrift  fur  physiologische  Chemie, 
1903,  XXXIX.,  p.  155. 

n  Osborne,  Leavenworth  and  Brautlecht,  Amer- 
ican Journal  of  Physiology,  1908,  XXIIL,  p.  180. 

"Fischer  and  Abderhalden,  Zeitschrift  fur 
physiologische  Chemie,  1904,  XLII.,  p.  540. 

13  Osborne  and  Harris,  Journal  American  Chem- 
ical Society,  1903,  XXV.,  p.  323. 

14  Hammarsten,  Zeitschrift  fur  physiologische 
Chemie,  1883,  VII.,  p.  227. 


8 


specific  immunity  reactions.  The  published 
feeding  experiments  in  which  a  single  purified 
protein  has  been  administered  to  animals  are 
all  limited  in  their  duration  to  periods  of  days 
or  weeks  which  are  too  brief  to  furnish  con- 
vincing data.  Indeed,  one  will  scan  the  lit- 
erature in  vain  for  properly  controlled  experi- 
ments in  which  isolated  and  purified  proteins 
have  been  fed  successfully.15 

Without  citing  here  the  numerous  failures 
and  the  successive  changes  instituted  in  our 
earlier  trials,  we  may  briefly  call  attention  to 
some  of  the  purely  "  nutritive  "  factors  which 
have  had  to  be  taken  into  consideration.  The 
energy  requirement  must  obviously  be  satisfied 
in  an  available  form.  A  minimum  protein 
requirement  must  likewise  be  provided  in  any 
event.  Experiments  which  are  to  continue 
over  more  than  very  few  days  must  include  a 
suitable  quota  of  inorganic  salts — so-called 
mineral  nutrients.  This  is  in  itself  a  problem 
of  fundamental  importance,  the  study  of 
which  has  barely  been  begun  in  any  synthetic 
way.  One  may,  it  is  true,  imitate  the  "  ash  " 
of  milk  or  blood;  but  the  elements  occur  here 
in  combinations  quite  different  from  those 
prevailing  in  the  tissue  fluids  themselves  or  in 
the  native  foods.  The  balance  of  acid  and 
basic  groups,  the  changing  need  for  individual 
elements  like  phosphorus,  calcium,  chlorine 
and  iron,  furnish  a  series  of  complex  variables 
which  are  probably  as  indispensable  to  certain 
aspects  of  nutrition  as  they  are  unappreciated. 
If  to  all  this  is  added  the  uncertain  signifi- 
cance of  the  as  yet  largely  unidentified  com- 
pounds such  as  cholesterol  and  phosphatides 
which  occur  in  all  natural  food  mixtures,  the 
experimental  difficulties  begin  to  appear  in 
their  true  light. 

At  the  outset  it  is  only  fair  to  remark  that 

16  Cf.  Osborne  and  Mendel,  Publication  156, 
Part  L,  Carnegie  Institution  of  Washington,  1911, 
for  the  literature  on  these  topics. 


9 


a  successful  feeding  experiment  with  isolated 
food  mixtures  is  of  greater  import  than  a 


V 





Food  e 

iten 

 50- 

X 

-A  100 



1  dead 

Nitrofre 

1  balance 

jJtr- 

^4 

] 

Days 

Fig.  1.  (Taken  from  Carnegie  Publication  No. 
156,  page  26.)  Showing  the  continued  decline  of 
a  rat  on  a  dogbiscuit-lard  diet  for  103  days. 


failure  may  be;  for  ill  health  may  be  occa- 
sioned by  incidents  quite  apart  from  those 
already  outlined.  Accident  and  acquired  dis- 
ease, unrecognized  or  uncontrollable,  enter  into 
the  life  of  every  individual  and  serve  to  upset 
an  otherwise  normal  nutritive  equilibrium. 

Turning  to  the  present  experiments,  our 
earlier  attempts  were  largely  based  on  those 
of  our  predecessors.  Comparative  trials  with 
food  mixtures  precisely  alike  except  for  both 
content  and  character  of  the  inorganic  in- 
gredients soon  showed  the  great  importance  of 
this  feature.  A  fairly  suitable  salt  mixture 
was  thus  empirically  selected.  The  table  be- 
low may  serve  to  illustrate  the  character  of  the 
earlier  food  mixtures  which  experience  showed 
to  be  most  suitable. 

In  such  a  mixture  the  protein  can  be  varied 
without  serious  change  in  the  fuel  value. 
With  one  protein,  viz.,  zein  from  maize,  nu- 
tritive decline  was  apparent  from  the  outset. 


10 


The  failure,  as  actual  investigation  showed, 
can  not  be  attributed  solely  to  poor  utilization. 
With  all  the  other  proteins,  such  as  casein, 
legumin,  edestin,  glutenin  or  gliadin,  in  the 
mixtures  indicated,  grown  rats  have  been 
maintained  in  body  weight  for  much  longer 


\  Bod 

^ — \ 

r 

f 

V 

| 

fooi 

HM 

\ 

d 

Tl- 

t— ri 

i 

1 

Hi'.rcje 

A 

balance 

[rn 

-rf 

♦  0.05 

o 

£ 

<D 

•  -0.I0 


Oays 


Fig.  2.  (Taken  from  Carnegie  Publication  No. 
156,  page  42.)  This  rat  was  fed  for  169  days  on 
a  diet  containing  pure  casein  as  the  only  protein. 


Per  Cent. 


Isolated  protein    18 

Cane  sugar    15 

Starch    29.5 

Lard    30 

Agar-agar18    5 

Salt  mixture    2.5 


100.0 

16  This  indigestible  carbohydrate  was  added  to 
furnish  "  roughage' '  in  the  diet.  Cf.  Mendel, 
Zentrdlblatt  fur  Stoffwechsel,  1908,  No.  17; 
Swartz,  "Nutrition  Investigations  on  the  Carbo- 
hydrates of  Lichens,  Alga?,  and  Belated  Sub- 
stances, ' '  Transactions  Connecticut  Academy  of 
Arts  and  Sciences,  1911,  XVI.,  pp.  247-382. 


LI 


periods  than  we  have  found  recorded  by  pre- 
vious investigators. 

From  many  protocols  we  present  three  in 
graphic  form;  the  first  to  illustrate  a  failure 


r 

— y 

-y>  

 e 

I 
1 

v 

\ 

> 

1 

( 

f 

) 

0) 

o 

* 

c  , 
•2  \ 

) 
1 

1 

j5 
c 

r 

■I— 

1 

1 

i — j_ 

■A 

h 

-t--- 

_  n 

c± 

r 

1 

L 

1 

P  o 


cS   £  S 


from  the  outset,  the  second  and  third  as  ex- 
amples of  a  relatively  successful  attempt  over 
a  period  of  169  days  and  259  days,  respectively. 


12 


In  every  case — and  we  might  cite  very  many 
such  experiments  under  varied  conditions — a 
decline  ultimately  ensued  leading  to  death 
unless  a  dietary  change  was  instituted. 

It  early  seemed  unlikely  that  the  protein 
was  responsible  for  failures  of  this  character; 
for  this  foodstuff  forms  so  large  an  essential 


A     Body  weigh! 

 ^ 

2 

Food 

eaten 

\  / 

y 

— i.  _  ... 

.  A 

\\ 

 vvr~ 

! 

\  / 

\ 

\  / 
\  / 

V 

\ — ' — x 

\ 

\ 

!  ' — 

.r 

u-r 

Nitrogor 

balance 

-J" 

rfl- 

!  ' 

0  20  40  £0  20  100  120  1^0  ISO  :20  200  220  240 


Fig.  4.  (Taken  from  Carnegie  Publication  No.  156,  page  45.)  This  rat  was  fed  210  da 
on  a  diet  containing  casein  and  excelsin  as  the  only  proteins. 


component  in  the  mixture  that  a  defect  ought 
soon  to  manifest  itself — as  indeed  it  did  with 
zein.  Nor  did  the  animals  fare  better  when 
more  than  one  protein  was  present.  Here,  too, 
the  ultimate  decline  in  the  grown  rats  in- 
evitably showed  up,  as  will  be  seen  in  the 
illustrative  charts  below. 

Remarkable  in  this  connection  were  the  ob- 
servations made  on  small  white  rats  during 
the  period  of  active  growth.  Lacking  food,  at 
this  stage,  the  animals  speedily  die,  since  the 
reserve  stores  are  small  or  wanting.  With  an 
appropriate  mixed  diet  growth  is  vigorous,  and 
the  rate  of  gain  is  strikingly  similar  in  healthy 


13 


animals  of  related  origin.  When  young  rats 
are  fed  on  diets  containing  a  single  protein  in 
the  mixtures  described  above  they  fail  to  grow, 
although  they  can  be  maintained  at  uniform 
body  weight  and  size  for  long  periods.  Here 
then  is  an  evident  distinction  between  main- 
tenance and  growth  in  respect  to  the  function 
of  the  ration.  An  illustration  of  the  stunting 
of  animals  in  this  manner  is  graphically  af- 
forded by  the  appended  curves  in  which  a 
dwarfed  animal  is  compared  with  a  suitably 
fed  one  from  the  same  litter. 


2  

Body 

weight 

dead 

— V  

Food 

eaten 

1  rl 

-n-r 

Nitrcger 

balance 

1 

O  20  40  60  80  100  120  1*0  160 

Doys 


Fig.  5.  (Taken  from  Carnegie  Publication  No. 
156,  page  46.)  This  rat  was  fed  160  days  on  a 
diet  containing  casein  and  pea  legumin  as  the  only 
proteins. 

What  is  the  factor  or  what  are  the  causes 
connected  with  the  ultimate  failure  of  the 
older  rats  to  thrive  on  the  dietaries  outlined, 
or  of  young  rats  to  grow?  Evidence  which 
need  not  be  reviewed  here  pointed  to  some- 
thing other  than  the  character  of  the  protein, 
fat  or  carbohydrate.  Animals  will  thrive  and 
grow  on  a  "mixed"  diet  of  corn,  vegetables, 


14 


etc.;  but  we  have,  furthermore,  noted  that 
their  nutritive  needs  can  be  met  with  an  "  arti- 
ficial "  food  mixture  in  which  dried  milk  and 
fat  form  the  sole  ingredients.17     That  the 


Fig.  6.  Showing  the  body  weight  and  food  in- 
take of  a  small  rat  grown  normally  on  a  diet  of 
dried  milk  and  lard  in  the  upper  curves.  The 
lower  curves  are  charted  from  a  rat  of  the  same 
litter  maintained  without  growth  on  a  diet  con- 
taining glutenin  as  the  sole  protein  in  a  mixture 
unsuitable  for  growth. 

17  The  dried  milk  used  is  the  commercial  "Tru- 
milk, "  furnished  by  the  Merrell-Soule  Co.,  of 

Syracuse,  N.  Y. 


16 


stunted  or  malnourished  rats  in  these  earlier 
experiments  have  not  lost  their  capacity  to 
groiv  or,  in  the  case  of  the  adults,  have  not 
become  permanently  disorganized  from  a  nu- 
tritive standpoint  can  be  readily  demon- 
strated ;  for  they  will  resume  growth  or  become 
realiirented,  as  the  case  may  be,  as  soon  as 


14© 


4X 


i 


2*0 


'A 


lie 


no 


foot 


So- 

t5 


S  -J(  •--       -K  k  . 


BE 


3'* 


tt 


,  - 


rn 


Fig.  7.  Showing  the  real i mentation  of  a  rat  practically  moribund,  by  the  addition  of 
'otein-free  milk  to  the  diet  containing  a  single  protein  (casein). 


mixed  food  is  furnished.  The  milk  mixtures 
are  as  efficient  as  mixed  food  in  promoting 
growth  and  restoring  nutritive  equilibrium. 

Rats  have  been  carried  through  two  genera- 
tions on  a  food  mixture  of  the  following  com- 
position : 


1(3 


Per  Cent 

Milk  powder    60.0 

Starch    15.7 

Salt  mixture    1.0 

Lard    23.3 

100.0 

Obviously  the  milk  contains  the  nutrient 
elements  essential  to  success  which  had  previ- 
ously not  been  satisfactorily  imitated  in  the 
artificial  food  mixtures.  It  occurred  to  us  to 
attempt  to  locate  these  as  yet  unknown  com- 
ponents by  removal  of  the  proteins  from  milk 
and  concentration  of  the  protein-free  (and  fat- 
free)  residues.  The  product  thus  obtained 
(and  which  may  conveniently  be  termed  "  pro- 
tein-free milk " 1S)  has  fulfilled  our  expecta- 
tion and  enabled  us  at  length  to  study  the 
relative  value  of  added  proteins  in  the  dietary. 
The  protein-free  milk  contains  the  milk  sugar 
in  addition  to  inorganic  salts  and  other  as  yet 
unknown  components  of  the  milk.  Whether 
it  is  the  peculiar  combinations  of  the  latter, 
or  some  ideal  "  balancing "  of  the  inorganic 
ions  therein,  or  the  presence  of  traces  of  essen- 
tial organic  compounds,  or  all  of  these,  which 
guarantee  the  successful  outcome,  remains  to 
be  ascertained. 

What  has  been  accomplished  thus  far  with 
the  new  possibilities  of  investigation  at  hand 
may  be  mentioned  in  brief.  Rats  which  have 
developed  marked  symptoms  of  decline  on 
mixtures  of  isolated  food  substances  contain- 
ing a  single  protein  have  been  revived  in  a 
way  little  short  of  marvelous  by  the  substitu- 
tion of  the  protein-free  milk  in  place  of  part 
of  the  previous  (non-protein)  food.  Instances 
have  occurred  where  successful  realimentation 
has  thus  followed  in  animals  practically  mori- 
bund. The  chart  below  furnishes  a  graphic 
illustration. 

18  A  detailed  description  of  the  preparation  and 
composition  of  protein-free  milk  is  given  in  the 
detailed  papers,  Part  II. 


SI 


4- 


.g 

£  s 
Pi  1=1 


3LC 


.9  .9 
§  & 


^4 


43  fl 
be  2 

O  M 

►•a 

"8  & 


ft 


5K 


•V 


111 

S       O  Pi 

00  ■§  ? 
ad  *  I 

.  pi 

©  2  *h 

H  O 

bo  -3 

O  C3 

-M  pi 


18 


Even  more  interesting  is  the  role  of  this 
protein-free  milk  in  facilitating  growth.  By 
the  use  of  protein-free  milk  to  furnish  the 
"  accessory  "  portions  of  the  diet  the  relative 
deportment  of  different  proteins  in  growth  has 
been  investigated.  Thus  adequate  growth  has 
been  noted  where  the  sole  protein  was  either 
the  casein  of  milk,  the  lactalbumin  of  milk, 
crystallized  egg  albumin,  crystallized  edestin 
from  hempseed,  the  glutenin  of  wheat,  or  gly- 
cinin  from  the  soy  bean.  But  not  all  proteins 
suffice  to  promote  growth  under  otherwise 
favorable  conditions.  The  gliadin  of  wheat 
(notably  lacking  in  glycocoll  and  lysine)  and 
the  hordein  of  barley  (closely  resembling 
gliadin  in  its  chemical  constitution)  suffice 
for  maintenance  without  growth.  Zein,  the 
tryptophane-,  lysine-  and  glycocoll-free  protein 
of  maize,  is  alone  insufficient  for  the  main- 
tenance requirement.  How  well  growth  can 
proceed  under  these  somewhat  artificial  condi- 
tions of  diet  is  shown  in  a  few  charts  in  which 
the  curve  of  growth  on  mixed  food  is  simul- 
taneously plotted. 

How  entirely  different  the  results  are  when 
an  "  inadequate  "  protein  is  alone  furnished, 
despite  an  abundant  ingestion  of  food,  is 
strikingly  shown  by  the  drawings.  The  ani- 
mals A  and  B  were  of  one  age  and  differed 
simply  in  having  been  sustained  on  different 
proteins. 

It  will  be  noted  that  the  older  but  stunted 
animals  do  not  vary  materially  in  size  from 
properly  nourished  younger  animals  which 
have  attained  the  same  body  weight.  Herein 
they  differ  essentially  from  young  animals 
which,  maintained  at  constant  body  weight  by 
underfeeding,  continue  to  grow  in  size.  Such 
conditions  have  been   described  in  cattle,1' 

"Waters,  Proceedings  Society  for  the  Promo- 
tion of  Agricultural  Science,  1908,  XXIX.,  p.  3; 
also  Ibid.,  XXX.,  p.  71. 


19 


dogs  20  and  children  ;21  and  they  lead  to  dispro- 
portioned  forms.  Our  (malnourished  rather 
than  undernourished)  rats  have  merely  main- 
tained themselves,  if  we  except  the  possibility 
of  a  continued  development  of  the  nervous 


c 

This  drawing  shows  the  influence  of  different 
proteins  on  growth.  A  and  B  are  rats  of  the  same 
brood  which  were  fed  from  the  time  of  weaning 
on  foods  of  the  same  composition  except  that  the 
diet  given  to  A  contained  pure  casein  while  that 
given  to  B  contained  pure  gliadin  as  its  only  pro- 
tein. The  appearance  of  B  at  the  age  of  140  days 
closely  resembles  that  of  C,  a  normally  nourished 
rat,  which  at  the  age  of  36  days  had  the  same 
weight  as  B.  (Sketch  from  photographs  in  Publi- 
cation 156,  Part  II.,  Carnegie  Institution  of  Wash- 
ington, 1911.) 

system  of  which  we  have  furnished  some  evi- 
dence elsewhere.22 

Aside  from  the  nutritive  inequalities  of  dif- 
ferent proteins,  as  well  as  the  apparent  com- 
parable suitability  of  chemically  and  biolog- 
ically unlike  proteins — all  of  which  remains  to 
be  subjected  to  more  refined  experimental  in- 

"Aron,  Biochemische  Zeitschrift,  1910,  XXX., 
p.  207 ;  Philippine  Journal  of  Science,  Sec.  B., 
1911,  VI.,  p.  1. 

21  Fleischner,  Archives  of  Pediatrics,  October, 
1906. 

22  Osborne  and  Mendel,  Carnegie  Institution  of 
Washington,  Publication  156,  Part  II. 


20 


vestigation — it  is  worth  while  to  point  out 
numerous  other  incidental  findings.  Animals 
which  have  grown  from  small  size,  e.  g.,  40 
grams,  to  adult  form,  e.  g.,  160  grams,  and 
have  thus  quadrupled  their  weight  on  a  diet 
furnishing  its  nitrogen  in  the  form  of  a  simple 
protein  like  edestin,  have  by  some  process  per- 
fected the  synthesis  of  purines  and  nucleo- 
proteins,  perchance  of  phosphoproteins  and 
nitrogenous  phosphatides,  and  of  ferruginous 
proteins  (like  hemoglobin)  from  iron-free  pro- 
tein precursors  and  "  inorganic  iron." 

With  what  powers  of  synthesis  in  such 
directions  is  the  body  provided  by  nature? 
What  modifications,  if  any,  can  be  introduced 
into  the  organism  in  respect  to  structure, 
function  or  inheritance  by  the  possibility  of  a 
successfully  regulated  control  of  the  character 
of  the  most  important  foodstuff,  the  protein? 
Such  physiological  and  broader  biological 
questions  appear  to  lend  themselves  to  experi- 
mental study  by  the  methods  which  we  have 
initiated.  There  are,  further,  pathological 
aspects  involving  abnormal  growth,  dwarfism, 
recuperation  and  senescence  which  similarly 
suggest  themselves.  The  program  for  the 
future  is  limited  only  by  the  success  and  effi- 
ciency of  the  methods  adopted. 

To  the  biological  chemist,  no  feature  of 
these  problems  appeals  more  strongly,  perhaps, 
than  the  question  of  how  an  organism  can 
build  such  diverse  nitrogenous  tissues  from  a 
single  dietary  protein.  It  is  true  that  the 
newer  conceptions  of  the  extensive  role  of 
hydrolysis  in  digestion  prior  to  absorption 
have  extended  the  inquiry  a  step  further,  so 
that  we  may  ask  what  is  the  minimum  of  this 
or  that  amino-acid  or  simple  polypeptide  re- 
quired. But  we  have  seen  rats  grow  for 
months  with  casein — thoroughly  purified  and 
glycocoll-free — as  the  sole  source  of  these 
amino-acids.  During  this  time,  one  animal 
even  brought  forth  two  broods  of  young  and 


21 


secreted  milk  in  sufficient  quantity  to  bring 
her  young  to  the  age  when  they  were  able  to 
care  for  themselves.  Another  pair  of  rats 
maintained  178  days  on  gliadin  as  the  sole 
protein  of  the  diet  produced  healthy  young  and 
successfully  reared  them.  It  is  most  unlikely 
from  all  that  is  otherwise  known,  that  the 
tissues  of  our  experimental  animals  are  chem- 
ically imperfect  or  essentially  unlike  those  of 
normally  fed  rats  which  presumably  do  contain 
glycocoll  and  lysine  groups.  Have  we  hereto- 
fore underrated  the  ultimate  synthetic  capac- 
ities of  animal  cells  % 28 

The  observation  that  animals  long  main- 
tained on  diets  of  the  character  used  in  our 
feeding  trials  voraciously  eat  the  feces  of 
normally  fed  rats  led  us  to  experiment  in 
another  direction.  It  has  been  noted  as  a 
result  of  this  that  in  a  not  inconsiderable 
number  of  instances  the  feeding  of  small  por- 
tions of  "  normal "  rat  feces  tended  to  check 
the  decline  of  rats  kept  on  pastes  of  isolated 
food  substances  containing  the  earlier  salt 
mixture.  The  possibility  of  altering  the 
bacterial  flora  of  the  alimentary  tract  by 
dietetic  conditions  at  once  suggests  itself 
in  this  connection,  and  reference  may  be  made 
to  the  significant  studies  of  Herter  and  Ken- 
dall,24 among  others,  which  elucidate  this  ques- 
tion. To  what  extent  is  the  cooperation  ©f 
bacteria  either  essential  or  useful  in  the  ali- 
mentary functions?  This  is,  indeed,  still  a 
debated  question.25    But  one  can  not  dispel  the 

23  One  is  reminded  of  the  recent  studies  of 
Knoop,  Zeitschrift  fur  physiologische  Chemie, 
1910,  LXVIL,  p.  489,  and  Embden  and  Schmitz, 
Biochemische  Zeitschrift,  1910,  XXIX,  p.  423, 
bearing  on  such  possibilities.  Cf.  Mendel,  Ergeb- 
nisse  der  Physiologie,  1912,  XI. 

24  Herter,  ' '  The  Common  Bacterial  Infections  of 
the  Digestive  Tract,"  The  Macmillan  Co.;  Herter 
and  Kendall,  Journal  of  Biological  Chemistry, 
1910,  VII.,  p.  203;  Kendall,  Journal  of  the  Amer- 
ican Medical  Association,  April  15,  1911. 


22 


idea  that  bacteria  might,  after  all,  enter  into 

reconstructive  reactions  which  may  furnish 
new  nitrogenous  complexes  from  amino-acids. 
Viewed  in  this  light,  the  immediate  hydrolysis 
products  of  our  foodstuffs  may  become  avail- 
able only  after  they  have  in  greater  or  less 
part  been  reconstructed  by  the  preeminently 
synthetic  symbiotic  bacteria  into  products  of 
more  uniform  character,  possibly  widely  dif- 
ferent from  the  original  intake.  Nucleopro- 
tein  synthesis,  for  example,  may  thus  become 
referable  to  bacterial  intervention;  and  the 
subtle  influence  of  the  indeterminable  non- 
protein factors  may  lie  in  some  measure  in  the 
regulation  which  they  exert  upon  the  micro- 
organisms of  the  gastro-intestinal  tract.2'  In 
any  event  such  suggestions  need  to  be  dealt 
with. 

It  is  hoped  to  continue  these  nutrition 
studies,  the  possible  scope  of  which  has  barely 
been  indicated  in  what  has  gone  before.  They 
seem  to  us  to  justify  the  effort  which  has  been 
involved.  Indeed  only  by  unremitting  re- 
gard for  details,  such  as  the  careful  purifica- 
tion and  preparation  of  the  materials  fed  and 
attention  to  the  animals,  can  the  uncertain 
factors  be  limited,  comparable  results  obtained 
and  definite  conclusions  safely  drawn.  We 
realize  that  only  a  beginning  has  been  made, 
and  believe  that  further  progress  is  possible. 

Thomas  B.  Osborne, 
Connecticut  Agricultural 
Experiment  Station 
Lafayette  B.  Mendel, 
Sheffield  Laboratory  of 
Physiological  Chemistry, 
Yale  University 

New  Haven,  Connecticut 

25  Nuttall  and  Thierf elder,  Zeitschrift  fur  physi- 
ologische  Chemie,  1895,  XXI.,  p.  109;  Sehottelius, 
Archiv  fiir  Hygiene,  1908,  67,  pp.  177-208. 

26  Cf.  Armsby,  "The  Nutritive  Value  of  the 
Non-protein  of  Feeding  Stuffs,"  Bureau  of  Ani- 
mal Industry,  Bulletin  139,  1911. 


Reprinted  from  The  Journal  of  Pharmacology  and  Experimental  Therapeutics 
Vol.  Ill,    No.  fi.   July,  1912 


THE  ACTION  OF  SALTS  OF  CHOLINE  ON  ARTERIAL 
BLOOD  PRESSURE 

LAFAYETTE  B.  MENDEL,  FRANK  P.  UNDERHILL,  and  R.  R.  RENSHAW 

From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University,  New 
Haven,  Connecticut,  and  the  Chemical  Laboratory  of  Wesleyan  University, 
Middletown,  Connecticut 

The  discovery  of  the  presence  of  choline  in  many  of  the  animal 
tissues  either  preformed  or  as  a  derivative  of  larger  complexes, 
and  the  possible  relationship  cf  this  base  to  some  of  the  so-called 
"  hormone"  effects  in  the  organism  has  lent  a  new  interest  to  the 
precise  determination  of  its  physiological  role.  The  contention 
that  absolutely  pure  choline  salts  fail  to  induce  the  characteristic 
fall  in  blood  pressure  commonly  described  as  a  typical  effect  of 
the  injection  of  this  compound  was  supported  several  years  ago 
by  Popielski  and  his  pupil  Modrakowski.1  They  believed  that 
pure  choline  causes  only  a  rise  of  pressure;  and  they  maintain  that 
those  investigators  who  report  a  fall  of  pressure  have  worked  with 
impure  or  deteriorated  preparations.  This  contradiction  of  the 
customary  teaching  regarding  the  physiological  action  of  choline 
naturally  elicited  a  speedy  reinvestigation  of  the  question,  to 
which  we,  among  others,  contributed  a  preliminary  reply  in  1910. 2 
One  might  assume  that  our  experience,  together  with  the  experi- 
mental studies  of  Abderhalden  and  Muller,3  Lohmann,4  Berlin,5 

1  Modrakowski :  Pfliiger's  Archiv  fur  die  gesammte  Physiologie,  1908,  cxxiv, 
p.  601. 

2  Mendel  and  Underhill:  Zentralblatt  fur  Physiologie,  1910,  xxiv,  p.  251  (June 
25). 

3  Abderhalden  and  Muller:  Zeitschrift  fur  physiologische  Chemie,  1910,  lxv. 
p.  420;  1911,  lxxiv,  p.  253. 

*  Lohmann:  Zeitschrift  fur  Biologie,  1911,  lvi,  p.  1. 
6  Berlin:  Zeitschrift  fur  Biologie,  1911,  lvii,  p.  1. 

649 


650         L.  B.  MENDEL,  F.  P.  UNDERHILL  AND  R.  R.  RENSHAW 

Hunt  and  Taveau6 — to  mention  only  a  part  of  those  which  have 
arisen — had  adequately  controverted  the  newer  claims  and  re- 
stored confidence  in  the  earlier  view  point.  However,  the  con- 
tinued reiteration  of  Popielski's  allegations  in  recent  papers7 
induces  us  to  present  a  few  further  details  of  our  trials.8  They 
were  carried  out  entirely  with  synthetic  preparations  made  by 
Dr.  Renshaw  at  Middletown  according  to  the  improved  method 
devised  by  him.9  Since  the  controversy  centers  largely  in  the 
purity  of  the  salts  used,  our  attention  has  been  devoted  primarily 
to  this  point.  We  have  been  unable  to  confirm  Popielski  and 
Modrakowski's  claims  respecting  the  absence  of  depressor  effects 
when  the  purity  of  the  choline  products  is  sufficiently  assured. 

PREPARATIONS  OF  CHOLINE  USED — METHODS 

Eight  different  preparations  of  choline  chloride  and  sulfate 
were  tested  in  numerous  trials.  The  animals  (usually  cats)  were 
maintained  in  deep  ether  anaesthesia.  The  importance  of  this 
deserves  the  emphasis  which  Abderhalden  and  Mliller  have  given 
to  it;  for  when  the  narcosis  is  not  adequate  it  is  easy  to  obtain 
transitory  rise  of  pressure  reflexly  by  even  slight  manipulation  of 
the  animal  prior  to  the  act  of  intravenous  injection.  Unless 
specifically  stated  otherwise,  the  choline  salts  were  dissolved  in 
0.9  per  cent  NaCl  solution  just  prior  to  the  injection  into  an 
exposed  vein.  Popielski  remarks :  "Die  Schwierigkeit  der  physiol- 
ogischen  Untersuchung  des  Cholins  ist  auf  die  grosse  Schwierigkeit 
der  Gewinnung  von  chemisch  reinen  Praeparaten  zuriickzufuh- 

6  Hunt  and  Taveau:  Bull.  No.  73.  Hygienic  Laboratory,  U.  S.  Pub.  Health  and 
Mar.  Hosp.  Service,  Washington,  1911,  p.  12.  Details  of  the  literature  will  be 
found  in  this  paper  and  in  the  contributions  of  Abderhalden  and  Miiller,  so  that 
they  need  not  be  repeated  here.  Cf.  also  Gautrelet:  Journal  de  physiologie,  1909, 
xi,  p.  227;  v.  Fiirth:  Probleme  der  physiologischen  und  pathologischen  Chemie, 
1912,  p.  186,  ff. 

7  Cf .  Studinski:  Archiv  fur  experimented  Pathologie  und  Pharmakologie, 
1911,  lxv,  p.  155  (Popielski's  laboratory);  also  Samelson:  ibid.,  1912,  lxvi,  p. 347. 

8  A  report  was  presented  to  the  American  Society  for  Pharmacology  and  Experi- 
mental Therapeutics  at  the  meeting  in  December,  1911.  Cf .  Journal  of  Experimen- 
tal Pharmacology  and  Therapeutics,  1912,  iii,  p.  457. 

9  Renshaw:  Journal  of  the  American  Chemical  Society,  1910,  xxxii,  p.  128;  Abcfer- 
halden's  Biochemisches  Handlexikon,  1911,  iv,  p.  829. 


CHOLINE  AND  BLOOD  PRESSURE 


651 


ren."10  Accordingly  we  shall  describe  some  of  our  products  in 
sufficient  detail  to  permit  the  reader  to  form  a  more  critical  judg- 
ment as  to  their  probable  purity. 

Choline  chloride:  Preparations  1,  2  and  3 

Preparation  1  was  the  product  of  the  sixth  reprecipitation  of 
an  alcoholic  solution  of  our  synthesized  material  with  ether.  Not 
more  than  one-third  of  the  dissolved  product  originally  used  was 
removed  from  the  solution,  thus  creating  ideal  conditions  for  the 
exclusion  of  soluble  impurities.    An  analysis  gave: 

Cl  found  25.45  per  cent,  25.49  per  cent;  calculated,  25.41  per  cent 

Preparation  2  was  obtained  after  five  additional  reprecipitations 
of  1. 

Preparation  3,  our  purest  specimen,  was  the  product  of  the 
fifteenth  reprecipitation. 

The  second  of  three  partial  precipitations  on  both  the  first  and 
the  second  solutions  of  the  choline  chloride  was  taken  as  the  sample 
for  purification.  From  the  third  on  the  procedure  was  as  indi- 
cated. The  reason  was,  of  course,  to  eliminate  the  possibility 
of  any  amount  of  a  somewhat  more  insoluble  product  always  pre- 
cipitating with  the  chloride.  Had  such  a  substance  been  present 
even  in  fair  amount  this  procedure  would  have  eliminated  all 
but  such  quantities  as  the  solvent  could  have  taken  care  of. 
Such  a  possibility  was  very  remote;  but  on  account  of  the  con- 
troversy it  seemed  worth  while  to  eliminate  every  point  that 
might  be  criticised.  None  of  these  preparations  had  any  odor 
of  trimethylamine,  nor  did  they  develop  it  rapidly  when  exposed 
to  sunlight.  This  is  important  in  relation  to  the  alleged  speedy 
deterioration  of  choline  salts  discussed  later.  In  view  of  the  differ- 
ences in  the  extent  of  purification  attempted  one  would  expect 
wide  variations  in  the  degree  of  purity  and  a  consequent  differ- 
ence in  physiological  action  dependent  upon  the  amount  of  con- 
taminating impurity;  i.e.,  preparation  3  ought  (according  to  the 

10  Popielski:  Zeitschrift  fur  physiologische  Chemie,  1910,  lxx,  p.  250. 


652 


CHOLINE  AND  BLOQJ)  PRESSURE 


653 


explanation  of  depressor  effects  on  the  impurity  hypothesis)  to 
be  less  active  physiologically  than  1  or  2. 

These  preparations  were  delivered  in  sealed  tubes  and  injected 
the  same  day.  Twenty-one  months  later  the  experiments  with 
these  products  were  duplicated,  the  preparations  having  been  kept 
meanwhile  in  glass-stoppered  bottles  in  a  dark  desiccator.  There 
is  no  evidence  for  any  lack  of,  or  quantitative  differences  in,  the 
depressor  effects  of  measured  doses  of  choline  chloride  1,  2  or  3; 
nor  has  the  long  time  interval  noticeably  altered  the  behavior  of 
the  purest  product,  3,  despite  Modrakowski's  contentions  of  the 
extreme  instability  of  choline  salts. 

The  illustrative  blood  pressure  tracings  should  be  read  from 
left  to  right.  A  few  of  the  numerous  data  are  summarized  in 
tabular  form  in  the  Appendix.  It  is  unnecessary  to  report  the 
observations  with  larger  doses,  since  they  were  not  essentially 
different  in  character.  Furthermore  the  fact  that  a  fall  was 
always  produced  even  with  the  very  small  doses  is  significant  of 
itself. 

Comparative  effects  of  choline  chloride  1  (recrystallized  six  times) 
and  3  (recrystallized  fifteen  times)  on  blood  pressure.  The  prep- 
arations were  not  more  than  twenty-four  hours  old 

Further  evidence  of  the  failure  of  this  pure  product  to  develop 
any  extreme  toxicity  on  standing  was  shown  by  experiments  made 
with  choline  chloride  3.  The  solution  was  prepared  one  month 
after  the  preparation  of  the  salt  and  then  allowed  to  stand  thirty- 
two  days  in  the  laboratory  before  being  tested.  Surely  here  was 
abundant  opportunity  for  the  development  of  the  extreme  de- 
pressor effects  assumed  to  be  characteristic  of  old  preparations 
and  depicted  in  the  curves  of  other  investigators  for  "  crude" 
choline.  The  tracing  shows  merely  the  typical  transitory  fall 
regularly  found  when  the  same  product  was  used  immediately 
after  its  delivery. 


654         L.  B.  MENDEL,  F.  P.  UNDERHILL  AND  R.  R.  RENSHAW 

Choline  chloride  3 — old  solution 

This  curve  may  also  be  compared  with  that  obtained  in  1910 
with  the  fresh  preparation.  (Cf.  Zentralblatt  fur  Physiologie, 
1910,  xxiv,  p.  252.) 


Cat.    2.8  kgm.    Injection  of  2.4  mgm.    (0.8  cc.) 
The  solution  stood  one  month  in  the  laboratory  before  being  used. 


Similar  depressor  results  were  obtained  on  dogs  with  the  fresh 
purest  choline  chloride  3,  even  with  doses  of  0.1  mgm.  per  kilo- 
gram.   The  salt  was  used  within  thirty  hours  after  it  was  made. 


CHOLINE  AND  BLOOD  PRESSURE 


655 


Choline  sulfate 

A  pure  preparation  of  this  salt11  was  likewise  examined.  The 
depressor  action  was  never  missed  even  in  small  doses.  It  was 
not  found  increased  when  the  same  preparation,  carefully  pre- 
served, was  tested  three  months  later;  i.e.,  there  was  no  evi- 
dence of  a  production  of  the  hypothetical  depressor  derivative  on 
standing. 

Since  Modrakowski  has  especially  emphasized  the  method  of 
Gulewitsch12  as  essential  to  obtain  pure  choline  we  have  also 
followed  this  plan  of  purification. 

A  solution  of  choline  chloride  was  evaporated  until  no  amine 
odor  was  detectable.  It  was  converted  into  the  platinic  salt, 
recrystallized  five  times,  decomposed  with  hydrogen  sulfide,  and 
finally  obtained  as  a  pure  crystalline  chloride  =  choline  chloride  4. 

This  salt,  injected  within  a  day  after  its  preparation  gave  the 
usual  characteristic  fall  of  pressure,  as  reported  likewise  by  Abder- 
halden  and  Mtiller  and  by  Lohmann  for  products  similarly  purified. 

Inasmuch  as  the  hypothetical  "impurities"  which  are  alleged 
to  account  for  the  fall  in  pressure  are  assumed  to  be  eliminated  by 
this  process  of  purification  we  searched  for  them  in'  the  wash 
solutions  or  mother  liquors  of  choline  chloride  4.  They  were 
precipitated  with  ether.  The  new  product,  choline  chloride  5 
in  which  the  decomposition  product  might  be  concentrated, 
might  be  expected  to  show  the  depressor  effects  in  marked  degree. 
This  was,  however,  not  the  case.  The  result  was  merely  the  typi- 
cal transitory  fall  of  arterial  pressure  characteristic  of  similar 
doses  of  the  purest  choline  salts. 

It  occurred  to  us  that  possibly  the  manipulations  to  which 
Modrakowski  subjected  his  solutions — the  evaporations  to  remove 
the  amine  odor,  the  treatment  with  hydrogen  sulfide,  a  possible 
failure  to  remove  the  last  traces  of  platinic  sulfide  which  separate 
with  difficulty — might  have  developed  some  antagonistic  sub- 
stance and  thus  "  masked"  the  typical  depressor  effect  despite  the 

11  Cf.  Renshaw:  Journal  of  the  American  Chemical  Society,  1910,  xxxii,  p.  129. 

12  Gulewitsch :  Zeitschrift  fur  physiologische  Chemie,  1898,  xxiv,  p.  513;  Cf. 
Modrakowski:  Pfliiger's  Archiv  fur  die  gesammte  Physilogie,  1908,  cxxiv,  p.  619. 


656         L.  B.  MENDEL,  F.  P.  UNDERHILL  AND  R.  R.  RENSHAW 

chemical  purification  of  the  choline  salts.  Solutions  of  our  prep- 
arations were  accordingly  evaporated  and  treated  with  hydrogen 
sulfide  alone  without  altering  the  physiological  effects  obtained. 
One  solution  was  evaporated  to  remove  the  amine  odor  com- 
pletely, recrystallized,  redissolved  in  alcohol,  and  reprecipitated 
with  ether  five  times.  A  blood  pressure  tracing  of  this  choline 
chloride  6  made  within  twenty-four  hours  is  reproduced  here. 


Cat.    2.8  kgm.    Injection  of  0.5  mgm.    (0.5  cc.) 


Choline  chloride  6 — repeatedly  purified 

That  traces  of  platinic  salts  do  not  alter  the  results  and  thus 
account  for  the  alleged  rise  of  pressure  was  plainly  shown  by 
numerous  trials  with  varying  proportions  of  the  heavy  metal 
added. 


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658         L.  B.  MENDEL,  F.  P.  UNDERHILL  AND  R.  R.  RENSHAW 


OTHER  FACTORS 

Modrakowski  has  maintained  (p.  620)  that  the  characteristic 
blood  pressure  effect  of  pure  choline  is  precisely  comparable  with 
that  obtained  from  "  impure"  preparations  after  atropine  is 
administered.  We  have  found  that  atropine  always  abolishes  the 
depressor  effects  of  choline  salts;  but  according  to  our  experience 
a  rise  in  pressure  under  these  conditions  is  only  obtained  when 
the  doses  of  choline  salts  administered  are  relatively  large.  This 
is  shown  in  the  appended  tracings. 

Choline  sulfate  following  atropine 

Section  of  the  vagi  does  not  alter  the  effect  of  pure  choline  salts. 

It  is  only  fair  to  point  out  that  our  preparations  do  not  exhibit 
any  of  the  extreme  depressor  effects,  with  attendant  symptoms, 
which  have  sometimes  been  described  by  those  who  have  used 
crude  commercial  products.  Modrakowski  has  pointed  out,  for 
example,  that  the  latter  in  doses  of  1  to  3  mgm.  per  kilogram  lead 
to  prolonged  inhibition  of  the  heart  beat  and  that  repeated  injec- 
tions may  impair  the  peripheral  endings  of  the  vagi  and  be  followed 
by  rise  in  pressure.  "Bei  rasch  hintereinander  erfolgenden  In- 
jektionen  vermag  die  blutdrucksteigernde  Cholinwirkung  doch 
durchzudringen"  (p.  620).  We  have  never  obtained  more  than  a 
transitory  fall  of  pressure  even  with  larger  doses  of  our  choline 
preparations  than  are  reported  in  this  paper.  On  the  other  hand 
a  rise  of  pressure  was  never  observed  (except  after  the  use  of 
atropine)  even  when  many  repeated  injections  were  undertaken. 

The  deterioration  of  choline  salts  on  standing 

Our  experience  throws  some  light  upon  this  question  which  is 
obviously  of  moment  in  the  controversy  at  hand.  In  pointing  out 
the  extraordinary  instability  of  even  pure  salts  of  choline  Modra- 
kowski wrote  (p.  622):  "Das  Kahlbaum'sche  Praparat  war,  wie 
bereits  erwahnt,  bei  seinem  Eintreffen  vollkommen  frei  von  Tri- 
methylamingeruch.  Gelegentlich  einer  kurzen  Unterhaltung  im 
Laboratorium  hielt  ich  dasselbe  einige  Augenblicke  in  der  Sonne; 
da  erregte  der  Umstehenden  und  meine  Aufmerksamkeit  ein 
deutlicher  Trimethylamingeruch,  welcher  der  halboff enen  Flasche, 


CHOLINE  AND  BLOOD  PRESSURE 


659 


die  die  Cholinchloridkrystalle  enthielt,  entstromte.  Es  zeigte 
sich  also,  dass  unter  geeigneten  Bedingungen  die  Zersetzung 
dieses  Praparates  fast  momentan  erfolgen  kann.  Die  Moglich- 
keit  erschien  daher  ausserst  wahrscheinlich  dass  kiirzere  oder  lan- 
gere  Aufbewahrung  die  Wirkung  des  Cholins  verandern  konnte." 
Lohmann,  on  the  other  hand,  insists  that  choline  chloride  does 
not  decompose  even  when  exposed  to  the  direct  sunlight  for 
months.13  This  is  in  accord  with  experience  of  Dr.  Renshaw  in 
similar  trials.  Abderhalden  and  Muller  are  uncertain  as  to 
whether  Kahlbaum's  preparations  are  contaminated  or  undergo 
secondary  decomposition.  Their  own  synthetic  chloride  gave 
no  odor  of  trimethylamine  after  being  preserved  sealed  in  the 
dark  for  six  months.  "Wir  mochten  uns  aus  diesen  Griinden 
vorlaufig  nicht  dariiber  aussern,  ob  ganz  reines  Cholinchlorhydrat, 
unter  geeigneten  Bedingungen  (verschlossen,  unbelichtet) ,  auf- 
bewahrt,  unbegrenzt  haltbar  ist."14  We  have  failed  to  note  a 
development  of  odor  after  two  years  in  preparations  thus  pre- 
served. In  any  event  no  deterioration  is  detectable,  as  already 
pointed  out,  from  the  standpoint  of  quantitative  alterations  in 
blood  pressure  effects  which,  after  all,  Modrakowski  used  as  his 
chief  criterion. 

CONCLUSIONS 

The  experiments  recorded  above,  along  with  numerous  further 
records,  afford  added  evidence  that  the  views  promulgated  by 
Popielski  and  his  pupils  about  the  physiological  behavior  of  salts 
of  choline  are  not  tenable.  Even  with  exceptionally  pure  syn- 
thetic salts  we  have  never  failed  to  observe  the  characteristic 
transitory  fall  of  arterial  pressure — a  fall  not  profound  or  pro- 
longed, but  never  absent  even  when  fractions  of  a  milligram  of 
purest  products  are  injected.  The  "  contamination"  theory  is 
rendered  improbable  by  the  fact  that  choline  salts  showed  no 
quantitative  differences  in  the  physiological  effect  when  different 
specimens  of  presumably  unequal  purity  were  investigated.  Fur- 
thermore, it  seems  extremely  doubtful  if  properly  prepared  and 
preserved  choline  salts  readily  decompose. 

13  Lohmann:  Zeitschrift  fur  Biologie,  1911,  lvi,  p.  16. 

14  Abderhalden  and  Muller:  Zeitschrift  fur  physiologische  Chemie,  1911,  lxxiv, 
p.  264. 


660         L.  B.  MENDEL,  F.  P.  UNDERBILL  AND  R.  R.  RENSHAW 


APPENDIX 

Selected  protocols  of  blood  pressure  experiments  with  salts  of  choline  (only  trials  with 
small  dosage  are  reported  here) 


PREPARATION  USED 

DOSE  PER 
KILOGRAM 

PALL  OF 
PRESSURE 
AFTER  THE 

REMARKS 

mgm. 

mm.  Hg. 

{  0.1 

4 

This  salt  was  recrystallized  six  times. 

Choline  chloride  1  

0.3 

26 

See  tracing  I 

0.5 

36 

0.1 

5 

This  salt  was  recrystallized  fifteen 

0.3 

22 

times.    See  tracing  I 

0.5 

30 

1.0 

24 

See  tracing  in  Zentbl.  Physiol.,  1910, 

xxiv,  252. 

Choline  chloride  3  

• 

0.23 

26 

After  being  preserved  twenty-one 

0.45 

36 

months  without  deterioration 

0.9 

26 

The  solution  stood  one  month  in  the 

laboratory  before  being  used.  See 

tracing  II 

0.1 

6 

Dog 

0.2 

28 

Doe 

Choline  chloride  4 

0.05 

15 

Purified  by  method  of  Gulewitsch 

Choline  chloride  5  

0.35 

16 

Fraction  which  ought  to  contain  the 

hypothetical  depressor  product  in 

marked  concentration 

Choline  chloride  6  

0.18 

24 

Repeatedly  purified.  See  tracing  III 

3.0 

30 

'  0.05 

11 

Choline  sulfate   < 

0.1 

17 

0.5 

32 

Reprinted  from  the  Proceedings  of  the  Society  for  Experimental  Biology  and  Medicine, 
1912,  ix,  pp.  123-124. 


87  (696) 

The  influence  of  tartrates  upon  phlorhizin  diabetes. 

By  FRANK  P.  UNDERBILL. 

[From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale 
University,  New  Haven,  Conn.] 

A  recent  communication  of  Baer  and  Blum  (Archiv  fur  Exper- 
imented Pathologie  und  Pharmakologie,  191 1,  65,  p.  1)  shows 
that  the  subcutaneous  administration  of  a  number  of  organic  com- 
pounds, containing  two  carboxyl  groups,  exercises  a  remarkable 
inhibitory  influence  upon  the  elimination  of  urinary  nitrogen  and 
dextrose  in  dogs  with  phlorhizin  diabetes.  Among  the  substances 
possessing  this  property  may  be  mentioned  glutaric  and  tartaric 
acids. 

In  an  endeavor  to  explain  the  mechanism  of  the  unique  influ- 
ence exerted  by  these  compounds  investigations  have  been  carried 
out  with  tartrates  upon  both  dogs  and  rabbits  under  conditions 
similar  to  those  established  by  Baer  and  Blum.  We  have  been 
able  to  corroborate  the  findings  of  Baer  and  Blum  with  respect 
to  the  action  of  tartrates  although  Ringer  (Proc.  Soc.  Exp.  Biol, 
and  Med.,  1912,  9,  p.  54)  failed  to  obtain  the  reported  results 
with  glutaric  acid. 

Our  interpretation  of  the  diminution  of  the  urinary  constituents 
is,  however,  entirely  different  from  that  offered  by  Baer  and  Blum. 
Tartrates  subcutaneously  injected  cause  a  prompt  disintegration 
of  the  cellular  elements  of  the  kidney  tubules,  leading  to  partial 
or  complete  loss  of  secretory  activity,  and  in  many  cases  to  anuria. 
Hence,  in  phlorhizin  diabetes  urinary  nitrogen  and  sugar  are  not 
eliminated  to  an  appreciable  extent. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  XI,  No.  1,  1912 


THE  HAEMAGGLUTINATING  AND  PRECIPITATING 
PROPERTIES  OF  THE  BEAN  {Phaseolus). 

By  EDWARD  C.  SCHNEIDER. 

(From  the  Department  of  Biology  of  Colorado  College,  Colorado  Springs, 

Colorado.1) 

(Received  for  publication  December  4,  1911.) 

The  extracts  of  a  number  of  kinds  of  seeds  are  capable  of  pro- 
ducing in  vitro  an  agglutination  and  sedimentation  of  the  red 
blood  corpuscles  of  various  animals.  This  peculiar  property  is 
largely  confined  to  species  of  the  Leguminosae  and  to  a  few  Sol- 
anaceae,  although  an  occasional  member  of  other  families  may 
possess  it.  The  property  was  first  noted  among  certain  toxic 
seeds;  the  several  species  of  Ricinus,  Abrus  pecatorius,  and  Croton 
tiglium.2  In  recent  years  the  list  has  been  enlarged  by  a  careful 
search  for  haemagglutinin  bearing  seeds.  Landsteiner  and  Raubit- 
schek3  found  this  property  in  extracts  of  beans,  Phaseolus,  peas, 
Pisum,  vetches,  Vicia,  and  lentils,  Ervum;  and  v.  Eisler  and  v. 
Portheim4  report  its  presence  in  five  species  of  Datura.  Mendel5 
added  the  following:  sweet  pea,  Lathyrus  odoratus;  lentil,  Lens 
esculenta;  yellow  locust,  Robinea  pseudacacia;  five  species  of  Vicia, 
Wistaria  Chinensis,  Caragana  arborescens;  senna,  Cassia  Mari- 
landica;  and  sweet  rocket,  Hesperis  malronalis.  He  also  found 
among  beans  that  the  haemagglutinins  are  absent  in  the  Lima 

1  Most  of  the  work  here  reported  was  done  in  the  Sheffield  Laboratory 
of  Physiological  Chemistry  of  Yale  University.  The  writer  wishes  to  ex- 
press his  hearty  thanks  to  Professor  Lafayette  B.  Mendel  for  the  sugges- 
tion of  the  problem  and  for  his  kindly  interest. 

2  For  the  early  literature  on  these  see  Jacoby:  Biochemische  Centralblatt. 
i,  p.  289,  1903. 

3  Landsteiner  and  Raubitschek:  Centralblatt  fur  Bakteriologie,  1  Abtei- 
lung,  xlv,  pp.  660-67,  1907. 

4  V.  Eisler  and  v.  Portheim:  Zeitschrift  fur  Immunitdtsforschung  und 
experimentelle  Therapie,  i,  p.  151,  1908. 

5  Mendel:  Archivio  di  fisiologia,  vii,  pp.  168-177,  1909. 

47 


48 


Haemagglutinin  of  the  Bean 


bean.  Wienhaus6  reports  that  this  property  occurs  in  the  soy 
bean,  Glycine  or  Soja  hispida;  and  Assmann7  found  it  is  the  seeds 
of  Canavalia  ensiformis,  Datura  stramonium,  and  three  species  of 
Lathyrus. 

The  agglutinative  property  is  not  necessarily  coincident  with  the 
toxic  activity  of  seeds.  It  varies  greatly  in  the  seeds  known  to 
contain  haemagglutinin  and  does  not  manifest  itself  equally  well 
with  the  blood  of  different  kinds  of  animals.  Among  laboratory 
animals  Mendel8  reports  the  blood  of  the  rabbit  to  be  most  sus- 
ceptible, and  those  of  the  pig  and  the  sheep  the  most  refractory. 
The  extract  of  a  number  of  the  seeds  noted  above  reacts  well  with 
rabbit's  blood  but  gives  negative  results  with  all  other  bloods 
tested.  The  reaction  is  strongest  with  suspensions  of  serum-free 
corpuscles.  Landsteiner9  found  the  normal  blood  serum  of  many 
kinds  of  blood  capable  of  checking  the  process  but  that  agglu- 
tination occurred  readily  when  washed  corpuscles  were  used. 

Several  workers  have  suggested  methods  for  obtaining  purified 
preparations  of  the  agglutinins  from  the  crude  extracts.  Land- 
steiner and  Raubitschek10  found  that  (1)  the  addition  of  a  little 
acid  produced  a  precipitate  which  contained  only  a  trace  of  the 
agglutinin,  the  chief  portion  remaining  in  the  filtrate.  (2)  When 
alcohol  was  added  an  agglutinative  precipitate  was  obtained. 
It  was  also  observed  that  when  this  precipitate  was  redissolved 
there  was  no  loss  of  power.  (3)  The  agglutinin  was  also  salted 
out  with  the  proteins  on  saturation  with  ammonium  sulphate. 

From  the  extract  of  beans  Wienhaus11  separated  a  mixture  of 
proteins  to  which  he  has  applied  the  name  of  Phasin.  Ten  grams 
of  bean  meal  were  extracted  with  500  grams  of  0.9  per  cent  sodium 
chloride  solution  for  twenty-four  hours  and  then  filtered.  To  the 
filtrate  an  equal  volume  of  alcohol  was  added.  A  voluminous 
precipitate  of  albumin  and  globulin  was  secured  in  which  the  agglu- 
tinin is  held  quantitatively.    On  drying  this  precipitate  in  a 

6  Wienhaus:  Biochemische  Zeitschrift,  xviii,  pp.  228-60,  1909. 

7  Assmann:  Pfluger's  Archiv,  cxxxvii,  pp.  489-510,  1911. 

8  Mendel :  Loc.  cit. 

9  See  Raubitschek:  Hamagglutinine  pflanzlicher.Provenienz  und  ihre  Anti- 
korper;  Kraus  and  Levadite's  Handbuch  der  Technik  und  Methodik  der 
Immunitatsforschung,  p.  625,  1911. 

10  Landsteiner  and  Raubitschek:  Loc.  cit. 

11  Wienhaus:  Loc.  cit. 


Edward  C.  Schneider 


49 


vacuum  he  secured  a  white  powder  which  yielded  to  physiological 
salt  solution  all  of  the  agglutinin  and  some  inactive  proteins.  He 
suggests  that  he  hopes  later  to  free  the  "Phasin"  from  proteins 
by  digestion. 

Landsteiner12  employed  the  characteristic  of  erythrocytes 
that  causes  them  to  give  up  to  the  suspension  fluid,  when  gently 
heated,  the  agglutinins  with  which  they  are  combined.  To  this 
end  he  agglutinated,  in  an  ice  chest,  sensitive  serum-free  corpus- 
cles with  purified  bean  extract  for  several  hours.  The  corpuscles 
were  then  washed  with  cold  isotonic  salt  solution  in  a  centrifuge 
until  no  trace  of  agglutinin  was  found  in  the  washing  solution.  The 
agglutinated  corpuscles  were  next  suspended  in  a  small  amount  of 
salt  solution  and  stirred  for  an  hour  at  45°  C.  With  precautions 
to  avoid  cooling  they  were  then  centrifugalized.  By  this  means 
he  obtained  a  clear  but  often  red  colored  solution  containing  the 
agglutinin.  This  he  found  he  could  further  purify  by  dialysis  or 
with  ammonium  sulphate. 

Thus  far  the  nature  of  these  vegetable  haemagglutinins  has  not 
been  satisfactorily  determined.  Landsteiner  and  Raubitschek 
conjecture  it  to  be  a  protein  by  analogy  with  the  very  pure  ricin 
isolated  by  Osborne,  Mendel,  and  Harris.13  The  latter  investi- 
gators separated  the  proteins  of  the  castor  bean,  Ricinus  Zanzi- 
bar ensis,  by  dialysis  and  fractional  precipitation  with  neutral  salts 
and  found  the  physiological  properties,  toxic  and  haemagglutina- 
tive,  to  be  associated  with  the  coagulable  albumin.  The  agglu- 
tinative action  was  absent  in  the  globulin  and  proteose  fractions, 
and  very  active  in  the  albumin  fractions. 

SEPARATION  OF  THE  PROTEIN  CONSTITUENTS  OF  THE  BEAN. 

In  view  of  the  experience  of  Osborne,  Mendel,  and  Harris  an 
attempt  has  been  made  to  separate  the  haemagglutinin  of  the 
Scarlet  Runner  bean,  Phaseolus  multiflorus,  Willd.  A  preliminary 
examination  of  a  number  of  varieties  of  beans  was  made  for  the 
purpose  of  determining  which  is  richest  in  haemagglutinins. 

12  See  Raubitschek  in  Kraus  and  Levadite's  Handbuch  der  Technik  und 
Methodik  der  Immunitdtsforschung,  p.  625,  1911. 

13  Osborne,  Mendel  and  Harris:  American  Journal  of  Physiology,  xiv,  pp. 
259-86, 1905. 

THE  JOURNAL  OP  BIOLOGICAL  CHEMISTRY,  XI,  NO.  1. 


5o 


Haemagglutinin  of  the  Bean 


Among  these  were  the  dwarf  wax-podded  varieties  Burpee's  Kid- 
ney, Wardell's  Kidney  Wax,  Red  Kidney,  Dwarf  Champion,  and 
Early  Six  Weeks;  and  the  climbing  wax-podded  variety  Golden 
Champion  of  P.  vulgaris,  L. ;  also  the  Scarlet  Runner,  P.  multi- 
florus,  Willd.  The  extracts  prepared  from  equal  weights  of  bean 
meal  were  almost  equally  active.  The  Scarlet  Runner  seed  is 
much  larger  than  the  seeds  of  the  other  varieties  which  favored  the 
removal  of  the  seed  coat. 

The  Scarlet  Runner  beans  were  first  passed  through  a  very  coarse  grinder. 
Much  of  the  seed  coat  was  thus  broken  away  from  the  substance  of  the  coty- 
ledons and  was  blown  out  with  an  air  blast.  Afterward  these  cracked  beans 
were  ground  to  a  coarse  meal  and  treated  with  benzine  to  remove  the  oil. 
Following  this  the  coarse  meal  was  ground  to  a  powder  and  1  kilo  of  it  was 
extracted  with  5  liters  of  a  2  per  cent  sodium  chloride  solution  that  had 
been  previously  heated  to  60°  C.  After  frequent  stirrings  for  two  hours  it 
was  placed  in  a  cold  room  over  night  and  then  filtered  perfectly  clear.  The 
extract  was  dialyzed  in  running  water  for  thirty-six  hours.    The  precipitate 

I  which  separated  was  filtered  from  the  solution  B  and  dried.  Unfortunately 
precipitate  1  was  dried  so  slowly  that  more  than  two-thirds  of  it  was  changed 
into  an  insoluble  protean. 

Solution  B  was  further  dialyzed  three  days  and  yielded  a  heavy  precipitate 

II  which,  when  dried,  was  more  than  85  per  cent  soluble.  The  solution  while 
dialyzing  tended  to  become  acid  in  reaction  and  required  frequent  neutrali- 
zation.   It  was  protected  against  decomposition  with  toluene. 

Solution  Bl,  which  remained  after  filtering  off  precipitate  11,  was  again 
dialyzed  four  more  days  and  yielded  a  small  amount  of  precipitate  111. 
Precipitates  1  and  11  were  the  globulin  phaseolin;  and  111  was  probably  the 
other  globulin,  phaselin,  separated  by  Osborne14  from  the  kidney  bean. 

The  proteins  remaining  in  solution  B2  (obtained  from  Bl  on  filtering  off 
precipitate  111)  were  salted  out  by  saturating  with  ammonium  sulphate. 
This  procedure  yielded  precipitate  IV  and  solution  B3.  Solution  B3  was 
then  dialyzed  in  running  water  until  free  from  salts  when  it  was  found  it 
did  not  contain  a  trace  of  the  haemagglutinin. 

Precipitate  IV  was  dissolved  in  a  small  volume  of  water  and  the  clear  solu- 
tion C  was  then  saturated  with  magnesium  sulphate  and  weakly  acidulated 
with  acetic  acid.  The  small  amount  of  precipitate  V,  which  will  be  called 
albumin,  was  then  filtered  from  solution  CI. 

The  albumin  precipitate,  V,  was  redissolved  and  precipitated  with  mag- 
nesium sulphate  and  then  dissolved  in  a  very  small  volume  of  water.  From 
this  solution  the  salt  was  removed  by  dialysis.  The  solution  was  next  evap- 
orated at  a  low  temperature  and  yielded  0.3  gram  of  albumin. 


14  Obsorne:  Journal  of  the  American  Chemical  Society,  xvi,  p.  635  and  p. 
707,  1894. 


Edward  C.  Schneider 


5i 


Solution  CI  was  dialyzed  for  several  weeks  until  free  from  salt,  then  was 
evaporated  in  low  dishes  at  48°  C.  Almost  a  gram  of  proteoses  was  secured 
from  this  solution. 

THE  ACTION  OF  THE  BEAN  PROTEIN  PREPARATIONS  ON  BLOOD. 

Tests  were  always  made  with  defibrinated  rabbit's  blood  diluted 
(1:5)  with  0.9  per  cent  sodium  chloride  solution.  One  cubic 
centimeter  of  this  blood  mixture  was  placed  in  a  small  and  very 
narrow  test  tube;  and  2  cc.  of  the  protein  preparation,  dissolved 
in  the  salt  solution,  were  added.  The  time  of  the  visible  beginning 
of  agglutination  and  the  condition  at  the  end  of  two,  four,  and 
twelve  hours  were  noted. 

Preliminary  tests  with  the  phaseolin  and  phaselin  (preparations 
I,  II,  and  III)  revealed  the  presence  of  haemagglutinin.  Prepara- 
tion II  was  most  active  but  none  of  the  globulin  preparations 
exhibited  the  property  in  a  striking  degree.  Believing  that  these 
proteins  adsorbed  the  haemagglutinin,  an  attempt  was  made  to 
purify  precipitate  II.  About  half  of  preparation  II  was  dissolved 
in  0.9  per  cent  sodium  chloride  solution.  One-half  of  this  solution 
was  dialyzed  until  it  yielded  its  phaseolin,  preparation  Ha,  and  the 
other  half  of  the  solution  was  saturated  with  magnesium  sulphate. 
The  resulting  precipitate  was  redissolved  in  water  and  on  dialysis 
yielded  preparation  lib.  Preparations  Ha  and  lib  were  less  active 
than  preparation  II.  This  weakening  in  activity  by  purification 
indicates  that  the  haemagglutinin  in  these  preparations  is  held 
there  by  adsorption. 

The  albumin,  purified  from  precipitate  V,  was  also  active  but 
less  so  than  the  globulins.  The  degree  of  activity  of  the  albumin 
and  globulins  is  given  in  Table  I. 

The  proteose  preparation  was  found  to  be  rich  in  haemagglu- 
tinin. It  produced  strong  agglutination  when  present  in  blood 
dilutions  of  one  part  to  100,000  and  more.  Wienhaus15  found  his 
crude  product  "Phasin"  completely  agglutinated  rabbit's  blood 
in  the  ratio  of  1:7000  in  fifteen  hours;  and  with  cat's  blood,  which 
reacted  still  better,  in  dilutions  of  1:11,000  in  eighteen  hours  and 
1 :60,000  in  twenty-three  hours.    Assmann16  also  working  with  a 

15  Wienhaus:  Biochemische  Zeitschrift,  xviii,  p.  232-33,  1909. 

16  Assmann:  Pfliiger's  Archiv,  cxxxvii,  pp.  489-510,  1911. 


52  Haemagglutinin  of  the  Bean 

TABLE  I. 


Agglutination  Trials  with  Protein  Preparations. 


PREPARATION  USED 

MILLIGRAMS 
ADDED  TO  1  CC. 
OF  BLOOD 
MIXTURE 

FIRST  INDICATION 
OF  AGGLUTINATION 

REMARKS 

No.  I.    Phaseolin ..  <j 

5.000 



2  minutes 

Complete  in  2  hours 

1.000 

25  minutes 

Partial  in  12  hours 

3.000 

At  once 

Complete  in  2  hours 

0.600 

2  minutes 

Complete  in  4  hours 

No.  II.    Phaseolin. . .  < 

0.300 

5  minutes 

Complete  in  12  hours 

0.200 

15  minutes 

Complete  in  18  hours 

0.150 

Trace 

0.100 

Negative 

0.600 

4  minutes 

Complete  in  4  hours 

No.  Ila.    Phaseolin  < 

0.300 

10  minutes 

Complete  in  12  hours 

0.200 

(?) 

Partial  in  12  hours 

0.150 

Negative 

1.500 

2  minutes 

Complete  in  12  hours 

No.  lib.    Phaseolin  .  - 

0.750 

(?) 

Complete  in  18  hours 

0.300 

Negative 

No.  III.    Phaselin..  .  J 

Complete  in  4  hours 

0.600 

(?) 

Trace 

No.  V.    Albumin.  .  .  .  j 

3.200 

4  minutes 

Complete  in  12  hours 

0.610 

N  egative 

0.300 

At  once 

Complete  in  2  hours 

0.060 

At  once 

Complete  in  2  hours 

Proteose  I 

0.020 

2  minutes 

Complete  in  4  hours 

0.015 

15  minutes 

Complete  in  12  hours 

0.0075 

19  minutes 

Complete  in  18  hours 

i  0.0037 

(?) 

Partial  in  18  hours 

"Phasin"  preparation  obtained  agglutination  of  diluted  rabbits 
blood  in  1 :35,Q00.  The  rapidity  of  action  and  the  dilutions  of  the 
proteose  preparation  that  are  effective  are  given  in  the  latter  part 
of  Table  I.  This  preparation  is  very  soluble  and  gives  the  dis- 
tinctive proteose  tests.  One  milligram  of  the  proteose  dissolved 
in  1  cc.  of  salt  solution  added  to  5  cc.  of  undiluted  defibrinated 
rabbit's  blood  produced  almost  instantaneous  agglutination;  and 
a  solid  clot-like  mass  of  corpuscles  settled  out  leaving  a  clear  serum 
in  less  than  half  an  hour.  Table  II  gives  further  testimony  as  to 
the  power  of  the  haemagglutinin  associated  with  the  proteose. 


Edward  C.  Schneider 


53 


TABLE  II. 


MILLIGRAMS  OF  PROTEOSE  ADDED 

VISIBLE 
AGGLUTINATION 

COMPLETE 
AGGLUTINATION 
AND 

SEDIMENTATION 

5  cc.  of  1:5  blood  < 

1.0  in  1  cc.  0.9  per  cent  NaCl 
0.5  in  1  cc.  0.9  per  cent  NaCl 
0.4  in  1  cc.  0.9  per  cent  NaCl 
0.3  in  1  cc.  0.9  per  cent  NaCl 
0.2  in  1  cc.  0.9  per  cent  NaCl 
0.1  in  1  cc.  0.9  per  cent  NaCl 

At  once 
At  once 
At  once 
At  once 
2  minutes 
4  minutes 

20  minutes 
45  minutes 

1  hour 

2  hours 
6  hours 

Incomplete 
in  6  hours* 

*  Further  observation  was  impossible  because  it  then  stood  over  night  at  room  temperature. 
All  but  the  last  gave  a  firm  clot-like  mass  in  the  time  recorded. 


It  seemed  probable,  in  view  of  the  observation  that  the  hemag- 
glutinin was  largely  confined  to  the  proteose  preparation,  that  all 
proteoses  might  cause  agglutination.  Hence  tests  were  made  with 
Witte's  peptone  upon  the  diluted  rabbit's  blood  corpuscles  but 
these  were  entirely  negative. 

DOES  AUTOLYSIS  ACCOUNT  FOR  THE  HAEMAGGLUTININ? 

The  presence  of  the  haemagglutinin  in  the  proteose  preparation 
also  suggested  that  it  might  be  a  product  of  the  hydrolysis  occur- 
ring in  the  solution  during  the  period  of  extraction  and  later.  To 
settle  this  point  a  fresh  extract  was  prepared  as  rapidly  as  possible 
and  immediately  tested  for  its  agglutinating  power.  A  portion 
of  the  extract  was  also  immediately  heated  for  five  minutes  at 
82°  C. — a  temperature  the  haemagglutinin  v/ithstands  for  thirty 
minutes  without  injury17 — the  coagulated  proteins  were  filtered 
off  and  the  filtrate  was  then  tested  for  the  relative  amount  of  haem- 
agglutinin. There  was  slightly  less  in  this  than  in  the  original 
extract  as  is  shown  in  Table  III.  This  is  very  likely  due  to  a 
slight  adsorption  by  the  coagulated  proteins.  The  remaining 
portion  of  the  original  extract,  after  the  addition  of  toluene,  was 
set  aside  in  a  cool  room  for  thirty  days.  Its  agglutinating  power 
was  again  determined  on  the  eighth  and  thirtieth  days.  There 
was  not  a  decided  change  in  agglutinating  power  as  will  be  observed 

17Wienhaus:  Loc.  ext. 


54 


Haemagglutinin  of  the  Bean 


TABLE  III. 


EXTRACT  DILUTED 
WITH  0.9  PER  CENT 

NaCl* 

FRESH  EXTRACT 

AFTER  HEATING 
FIVE  MINUTES  AT 

82°  C. 

EXTRACT  EIGHT 
DAYS  OLD 

EXTRACT 
THIRTY  DAYS 
OLD 

Undiluted 

Complete 

Complete 

Complete 

Complete 

1 

100 

Complete 

Complete 

Complete 

Complete 

1 

200 

Complete 

Complete 

Complete 

Complete 

1 

300 

Complete 

Complete 

Complete 

Complete 

1 

400 

Complete 

Partial 

Partial 

Complete 

1 

500 

Partial 

Slight 

Partial 

Complete 

1 

600 

Partial 

Negative 

Slight 

Partial 

*  Two  cubic  centimeters  of  extract  and  1  cc  of  1 :5  blood  used  in  each  test.  Agglutination 
recorded  at  end  of  twelve  hours. 


in  Table  III.  It  was  also  found  that  active  agglutinins  may  be 
secured  by  extracting  the  bean  meal  at  80°  C.  From  these  obser- 
vations it  would  seem  that  autolysis  does  not  account  for  the 
haemagglutinin  in  the  proteose  preparation. 

Digestion  trials.  Obsorne,  Mendel,  and  Harris18  showed  that 
the  toxicity  and  agglutinating  power  of  their  pure  preparation  of 
ricin  could  be  impaired  or  destroyed  by  pancreatic  digestion  pro- 
longed two  or  three  months.  Wienhaus,19  on  the  other  hand,  in 
digestive  trials  with  pepsin,  trypsin,  and  papain  made  on  his 
"Phasin"  for  periods  ranging  from  three  to  seven  days  failed  to 
show  any  destructive  action. 

The  haemagglutinative  proteose  preparation  was  subjected 
to  various  digestive  trials  with  trypsin,  erepsin,  and  mixtures  of 
these  two,  in  water  and  in  sodium  carbonate  solutions  for  a  period 
of  twenty-eight  days  with  practically  negative  results.  The  diges- 
tive mixtures  were  tested  with  fresh  blood  fibrin  and  Witte's 
peptone  several  times  during  the  period  and  found  to  be  active. 
Wienhaus  calls  attention  to  the  fact  that  his  "Phasin"  acts  as  a 
protein  and  he  expresses  the  opinion  that  it  is  a  protein  or  enzyme- 
like substance.  Since  Wienhaus'  digestion  trials  were  so  very 
short  and  the  effective  trials  of  Osborne,  Mendel,  and  Harris  were 
so  prolonged  the  failure  of  the  proteose  preparation  to  respond  to 
digestive  agents  in  the  time  allowed  still  leaves  the  question  of  the 

18  Osborne,  Mendel  and  Harris:  American  Journal  of  Physiology,  xiv,  p. 
284,  1905. 

19  Wienhaus:  Biochemische  Zeitschrift,  xviii,  p.  256,  1909. 


Edward  C.  Schneider 


55 


digestibility  of  these  haemagglutinins  open.  A  more  prolonged 
series  of  carefully  controlled  digestive  trials  is  planned  for  the  near 
future. 

IS  THE  HAEMAGGLUTININ  A  FOOD   STORED  FOR  THE  USE  OF  THE 
GROWING  SEEDLING? 

If  the  haemagglutinin  of  the  seed  is  a  proteose  it  should  readily 
be  utilized  by  the  growing  seedling  in  the  early  growth  after 
germination.  It  is  also  probable  that  preliminary  to  the  translo- 
cation of  the  protein  from  the  cotyledons  to  the  growing  tissues 
of  the  seedling  further  haemagglutinin  may  be  formed  from  the 
proteins  by  the  action  of  the  enzymes  evolved  during  germination. 
It  certainly  is  surprising  to  find  the  haemagglutinins  in  the  proteose 
portion  of  the  seed,  inasmuch  as  proteoses  and  peptones  are  not 
commonly  normal  constituents  among  the  reserve  proteins  of  seeds. 
They  are  of  course  present  to  some  extent  during  germination.  It 
may  be  noted  here  that  Osborne20  found  a  small  amount  of  pro- 
teoses when  he  studied  the  proteins  of  the  kidney  bean.  He  did 
not  determine  whether  the  proteoses  were  a  normal  constituent  of 
the  seed  or  a  product  of  autolysis  during  extraction.  Landsteiner 
and  Raubitschek21  showed  the  agglutinin  to  be  absent  from  green 
beans.  A  future  study  must  determine  when  the  haemagglutinin 
enters  the  seed  and  an  attempt  be  made  to  learn  its  source, 
whether  it  is  formed  in  the  seed  or  brought  to  it  to  be  stored. 

To  determine  if  the  haemagglutinin  is  utilized  by  the  seedling 
and  whether  it  is  increased  in  amount  during  germination  a  study 
was  made  of  seedlings  and  cotyledons  at  frequent  intervals,  from 
the  beginning  of  germination  until  the  depleted  cotyledons  fell 
from  the  seedling.  Two  series  of  observations  were  made,  one  with 
plants  grown  in  darkness  and  the  other  with  sturdy  plants  grown 
in  the  light.  For  the  determination  of  haemagglutinin  content 
the  seedlings  were  hastily  washed  and  the  cotyledons  separated 
from  the  seedling  close  to  the  stem,  and  then  cotyledons  and  seed- 
lings dried  separately.  When  dry  each  was  ground  to  a  powder 
and  known  weights  extracted  with  constant  proportions  of  a  0.9 

20  Osborne:  Journal  of  the  American  Chemical  Society,  xvi,  pp.  758-04, 
1894. 

21  Landsteiner  and  Raubitschek:  hoc.  cit. 


56 


Haemagglutinin  of  the  Bean 


per  cent  sodium  chloride  solution.  Three  kinds  of  beans  were  used, 
the  Scarlet  Runner,  Warden's  Kidney,  and  the  Early  Six  Weeks. 
The  cotyledons  of  the  last  two  are  lifted  above  the  soil  by  the  grow- 
ing stem  of  the  seedling  while  those  of  the  Scarlet  Runner  remain 
underground.  The  underground  habit  of  the  Scarlet  Runner  made 
it  difficult  to  secure  from  the  late  stages  cotyledons  that  had  not 
undergone  decomposition  to  some  extent.  The  data  obtained 
from  the  three  kinds  of  beans  were  wholly  concordant  through- 
out each  series. 

Repeated  tests  with  colorless  seedlings  and  with  the  green  leaves 
and  stems  of  those  grown  in  the  light  failed  to  show  the  slightest 
trace  of  agglutinative  power.  Hence  the  haemagglutinin  as  such 
is  not  carried  into  the  seedling  or,  at  least,  not  in  sufficient  amounts 
to  be  detected.  Roots,  stems,  and  leaves  were  also  examined 
separately  from  plants  of  many  sizes,  all  being  negative.  The 
agglutinin  is  not  a  normal  constituent  of  the  organs  of  the  vegeta- 
tive plant. 

As  a  type  of  the  results  obtained,  those  with  the  cotyledons  of 
the  Wardell's  Kidney  bean  are  given  in  Table  IV,  p.  57.  Dur- 
ing the  early  days  of  growth  the  agglutinative  action  for  equal 
weights  of  cotyledon  is  only  slightly  lowered,  which  indicates  that 
the  haemagglutinin s  are  withdrawn  gradually  along  with  other 
stored  foods.  Later  there  is  a  more  rapid  disappearance  of  the 
agglutinin.  Extracts  prepared  from  cotyledons  that  fell  from  the 
seedlings  of  Wardell's  Kidney  bean  and  Early  Six  Weeks  bean, 
grown  in  darkness,  gave  no  agglutinative  response.  From  seed- 
lings of  all  three  varieties  when  grown  in  light,  and  the  Scarlet 
Runners  grown  in  darkness,  it  was  impossible  to  get  depleted  coty- 
ledons wholly  free  from  the  haemagglutinins.  With  each,  however, 
there  was  a  very  marked  quantitative  reduction  in  this  property. 
It  follows,  therefore,  that  the  haemagglutinin  of  the  bean  is  utilized 
or  destroyed,  along  with  other  stored  foods,  by  the  developing 
seedling. 

THE  PRECIPITATING  REACTION  OF  BEAN  EXTRACTS. 

When  the  clear  extract  of  any  of  the  several  beans  examined  in 
this  study  was  added  to  rabbit's  blood  serum  a  flocculent  precipi- 
tate always  appeared.    The  reaction  usually  occurred  slowly. 


Edward  C.  Schneider 


57 


TABLE  IV. 

WardelVs  Kidney  Bean  During  Germination  and  Early  Growth. 


Grown  in  Light. 

WEIGHT  OF  TWENTY 

GREATEST  DILUTION  AT  WHICH 

COTYLEDONS 

LENGTH  OF  SEEDLING 

AGGLUTINATION  WAS  OBTAINED 

grams 

centimeters 

6.000 

o 

1:550 

0.918 

9.0 

1:350 

0.606 

13.2 

1:300 

0.300 

17.8 

1:100 

0.262 

* 

Undiluted 

Grown  in  Darkness. 

1.000 

9.4 

1:400 

0.508 

13.2 

1:200 

0.245 

18.3 

1:150 

0.215 

35.5 

Negative* 

*  Cotyledons  that  had  fallen  from  seedlings. 


For  some  minutes,  and  often  more  than  an  hour,  after  the  addition 
of  the  extract  to  the  blood  serum  the  mixture  remained  clear.  It 
then  gradually  became  cloudy  and  opaque,  finally  the  white  floccu- 
lent  precipitate  appeared.  The  entire  reaction  may  be  completed 
within  a  few  minutes  when  strong  extracts  are  used  but  will  require 
five  or  more  hours  with  dilute  extracts. 

This  precipitating  reaction  is  not  constantly  associated  with  the 
agglutinative  property  of  seed  extracts.  It  was  found  to  be 
absent  in  extracts  from  such  agglutinin  containing  seeds  as  the 
Wistaria  Chinensis,  the  hairy  vetch,  Vicia  vilosa,  and  the  pea, 
Pisum  sativum.  From  the  sweet  pea,  Lathyrus  odoratus,  an  extract 
was  obtained  that  gave  a  slight  clouding  of  the  serum  but  it 
failed  to  produce  a  precipitate. 

A  fresh  extract  prepared  from  Scarlet  Runner  bean  meal  was 
heated  repeatedly  at  various  temperatures  for  five-minute  inter- 
vals; and  after  each  period  of  heating  the  coagulated  proteins 
were  filtered  off  and  the  filtrate  then  tested  for  the  agglutinating 
and  precipitating  properties.  Both  properties  continued  practi- 
cally undiminished  up  to  a  temperature  of  80°  C.  At  83°  C.  the 
precipitating  power  was  destroyed  in  ten  minutes.    Table  V 


58 


Haemagglutinin  of  the  Bean 


TABLE  V. 


CONDITION  OF  EXTRACT 

PRECIPITATE  IN  SERUM 

AGGLUTINATION 
OF  CORPUSCLES 

Fresh  

Heavy  in  50  minutes 

moderate  in  4.5  hours 

Trace  in  7  hours 

Negative 

Negative 

Negative 

Negative 

Strong 
Strong 
Strong 
Strong 
Strong 
Strong 
Negative 

After  heating  at  80°  for  five  minutes. . 
After  heating  at  83°  for  five  minutes.  . 
After  heating  at  85°  for  five  minutes .  . 
After  heating  at  87°  for  five  minutes .  . 
After  heating  at  91°  for  five  minutes . . 
After  heating  at  94°  for  five  minutes . . 

shows  a  trace  present  after  five  minutes  at  83°.  The  agglutina- 
tive power  was  weakened  above  this  temperature,  but  withstood 
five  minute  exposures  to  91°,  and  was  wholly  destroyed  at  92°. 
Table  V  contains  the  data  obtained  from  one  series  of  heat  tests. 

The  protein  preparations  separated  for  the  study  of  the  agglu- 
tinins have  also  been  tested  for  the  precipitin  reaction.  The  glob- 
ulin preparations  II,  Ha,  and  lib  were  rich  in  it  while  I  contained 
a  trace.  The  albumin  (V)  gave  a  negative  test,  and  3  mgm.  of  the 
proteose  preparation  in  2  cc.  of  serum  failed  to  give  the  reaction. 

It  was  also  found  that  after  serum  had  been  added  repeatedly 
to  extract  until  no  more  precipitate  formed  that  the  mixture 
retained  its  agglutinating  power  practically  unaltered. 

These  several  differences  warrant  the  conclusion  that  the  precipi- 
tating and  agglutinating  properties  of  the  extracts  of  beans  are  due 
to  different  constituents  of  the  seed.  Or  we  may  better  express  it  that 
rabbit's  blood  contains  a  precipitin  for  certain  of  the  bean's  proteins. 

Wienhaus2*  made  certain  observations  which  are  of  interest  in 
this  connection.  He  found  his  "Phasin"  did  not  react  with  serum 
taken  from  hen's  blood.  On  adding  the  preparation  to  a  clear 
fluid  collected  from  the  joint  of  a  diseased  knee  a  heavy  precipi- 
tate was  obtained.  After  immunizing  rabbits  to  the  phasin  it 
was  impossible  to  obtain  a  precipitate  in  the  blood  serum  on  the 
addition  of  phasin.  He  points  out  that  this  is  contrary  to  the 
experience  of  Jacoby  and  others  when  they  immunized  animals  to 
ricin,  abrin,  and  crotin,  inasmuch  as  these  substances  gave  a  pre- 
cipitate when  added  to  the  immune  sera.  It  would  seem  from 
Wienhaus'  work  that  the  agglutinating  and  precipitating  proper- 
ties are  both  lost  for  the  blood  on  immunizing  the  animal. 

22  Wienhaus:  hoc.  ext. 


Edward  C.  Schneider 


59 


The  precipitating  property  does  not  occur  in  extracts  of  bean 
plants.  It  disappears  from  the  cotyledons,  as  does  the  agglutinin, 
with  germination  and  the  growth  of  the  seedling. 

SUMMARY. 

1.  The  proteose  prepared  from  the  Scarlet  Runner  bean  was 
found  to  be  a  very  active  haemagglutinating  agent.  Other  bean 
proteins  contained  some  haemagglutinin  but  this  was  shown  to  be 
adsorbed  by  them. 

2.  The  haemagglutinin  is  not  a  product  of  autolysis. 

3.  The  haemagglutinin  gradually  disappears  from  the  cotyle- 
dons, simultaneously  with  the  stored  food,  as  the  seedling  develops. 

4.  The  agglutinative  property  does  not  occur  in  the  extracts 
of  the  roots,  stems,  or  leaves  of  the  bean  plant. 

5.  The  addition  of  the  clear  extract  of  beans  to  rabbit's  blood 
serum  produces  a  flocculent  precipitate.  This  reaction  is  not  coin- 
cident with  the  agglutinating  property  of  all  haemagglutinin  con- 
taining seeds  and  appears  to  be  chiefly  associated  with  the  phaseo- 
lin  in  the  bean. 

Since  this  paper  was  written  there  has  been  brought  to  my 
attention  an  abstract,  in  the  Zentralblatt  fur  Biochemie,  xii,  p.  391, 
1911,  of  a  recent  paper  by  v.  Eisler  and  v.  Portheim.  They 
regard  the  haemagglutinin  as  a  protein  and  prove  it  to  be  a 
reserve  substance  that  disappears  from  the  embryo  during 
germination. 


THE  VALUE  OF  INULIN  AS  A  FOODSTUFF  * 


HOWARD  B.  LEWIS,  A.B. 

NEW  HAVEN,  CONN. 


For  many  years  physicians  have  sought  to  replace  the 
starch  in  the  diet  of  diabetics  by  some  other  carbohydrate 
which  is  well  tolerated  by  the  organism  and  which  can 
be  eaten  in  sufficiently  large  amounts  without  discomfort. 
Prominent  among  these  substitutes  for  starch  stands 
inulin,  a  carbohydrate  closely  resembling  starch  in  its 
physical  properties.1  Like  starch  it  exists  in  the  under- 
ground parts  of  many  plants,  as  a  reserve  carbohydrate, 
but,  unlike  starch,  it  is  a  polysaccharid  of  levulose,  the 
isomer  of  dextrose,  which  is  the  sugar  yielded  by  starch 
on  hydrolysis.  Inulin  occurs  in  the  roots  of  many  of  the 
Compositce,  particularly  in  the  tubers  of  the  dahlia,  the 
artichoke,  elecampane,  and  other  similar  plants.  Of 
these  vegetables,  the  artichoke  is  the  best  known  as  an 
article  of  diet,  and  is  most  frequently  recommended  for 
diabetics. 

Inulin  is  readily  hydrolyzed  to  levulose  by  dilute  acids, 
and  inasmuch  as  this  sugar  is  assumed  to  be  well  toler- 
ated by  most  diabetics,  inulin  appears  a  likely  source  of 
energy  for  the  organism.  Mendel2  has  pointed  out  that 
not  every  carbohydrate  by  virtue  of  its  chemical  com- 
position alone  is  a  true  nutrient  for  the  human  organism 
and  that  metabolism  experiments  with  a  study  of  the 
feces,  i.  e.,  a  determination  of  alimentary  digestibility, 
are  necessary  before  accepting  any  carbohydrate  as  an 
available  source  of  energy.  With  this  point  in  view,  the 
behavior  of  inulin  has  been  studied  in  a  healthy  man. 

As  early  as  in  1874,  Kiilz3  reported  that  no  sugar 
appeared  in  the  urine  of  diabetics  on  an  absolute  flesh 
diet  after  feeding  from  50  to  120  gm.  of  inulin.  No 

*  From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale 
University. 

1.  Von  Noorden  :  Von  Leyden's  Handbuch  der  Ernahrungsther- 
apie,  1904,  ii,  227. 

2.  Mendel  :  Zentralbl.  ges.  Physiol,  u.  Path,  des  Stoffwechsels, 
1908,  No.  17,  p.  1. 

3.  Kiilz  :  Beitriige  zur  Pathologie  und  Therapie  des  Diabetes 
Mellitus,  Marburg,  1874,  Jahresb.  Tierchem.,  1874,  iv,  448. 


2 


inulin  was  found  in  the  feces.  Hence  he  concluded  that 
inulin  is  completely  assimilated  in  both  mild  and  severe 
cases  of  diabetes.  Von  Mehring,  in  1876,4  reported  that 
the  use  of  inulin  did  not  increase  the  sugar  content  of 
the  urine.  More  recently  Teyxeira5  has  recommended 
the  addition  of  inulin  to  the  gluten  of  wheat.  Persia6 
stated  that  inulin  was  well  digested  and  assimilated  by 
diabetics  in  large  doses  and  through  long  periods.  He 
reported  that  the  feces  never  contained  large  amounts  of 
inulin.  Strauss7  reports  the  feeding  of  pure  inulin  with 
much  benefit  in  two  cases  of  diabetes.  After  admin- 
istering from  40  to  100  gm.  daily  he  found  no  sugar  in 
the  urine.  Strauss  recommends  the  use  of  vegetables 
rich  in  inulin,  such  as  artichokes  (14  per  cent,  inulin8). 
dandelions,  etc.  He  also  points  out  that  the  absorption 
of  inulin  naturally  proceeds  more  slowly  from  vegetables 
than  from  the  pure  substance.  Hale  White9  suggests 
that  the  tubers  of  the  dahlia  be  cooked  and  eaten  as  a 
vegetable  by  diabetics. 

The  failure  of  sugar  to  appear  in  the  urine  of  diabetics 
after  inulin  feeding  may  be  ascribed  to  a  tolerance  for 
levulose  formed  in  the  organism  by  the  conversion  of 
inulin.  But  it  is  also  possible  that  the  beneficial  effects, 
judged  from  the  standpoint  of  the  failure  of  sugar  to 
appear  in  the  urine,  may  be  due  to  the  fact  that  the 
inulin  is  not  utilized  and  hence  no  carbohydrate  is 
present  to  be  excreted  in  the  urine.  Inulin  may  be 
decomposed  in  the  intestinal  tract  or  pass  through  it 
unchanged  to  be  excreted  in  the  feces.  Miura10  and 
Mendel  and  Nakaseko11  have  shown  that  even  under  the 
most  favorable  conditions,  little  glycogen  is  formed  in 
rabbits  after  feeding  inulin.  Since  the  formation  of 
glycogen  takes  place  readily  after  levulose  feeding,  this 
strongly  indicates  a  lack  of  assimilation  of  inulin.  When 
inulin  is  introduced  parenterally  into  the  organism  there 
is  no  inversion  or  utilization,  the  inulin  being  excreted 
in  the  urine.12  Sandmeyer13  recovered  46  gm.  of  inulin 

4.  Von  Mehring  :  Beil.  Tagebl.  49  Versaml.  deutsch.  Naturforsch. 
u.  Arzte,  128  ;  Jahresb.  Tierchem.,  1876.  vi,  144. 

5.  Teyxeira  :  Boll.  chim.  Farm.,  xliii,  605-6  ;  Jahresb.  Tierchem., 
1905,  xxxv.  822. 

6.  Persia  :  Nuova  Revista  Clin.-Terapeut,  1905,  viii ;  Jahresb. 
Tierchem.,  1905,  xxv,  822. 

7.  Strauss  :   Therapie  der  Gegenwart,  1911.  lii,  337. 

8.  Allbutt  and  Rolleston  :  System  of  Medicine,  iii.  200. 

9.  Allbutt  and  Rolleston  :  System  of  Medicine,  iii,  204. 

10.  Miura  :  Ztschr.  f.  Biol.,  1905.  xxxii.  263. 

11.  Mendel  and  Nakaseko  :   Am.  Jour.  Physiol.,  1905,  xiv.  245. 

12.  Mendel  and  Mitchell  :   Am.  Jour.  Physiol.,  1905,  xiv,  239. 

13.  Sandmeyer  :  Ztschr.  Biol.,  1895,  xxxi,  32. 


3 


from  the  feces  of  a  depancreatlzed  dog  to  which  80  gm. 
had  been  fed. 

The  yiew  that  the  favorable  influence  of  inulin  feeding 
on  the  sugar  output  in  the  urine  is  the  result  of  non- 
utilization  due  to  non-absorption  is  confirmed  by  the 
work  of  Neubauer.14  In  a  case  of  levulosuria  he  found 
no  increased  levulose  content  of  the  urine  after  feeding 
80  gm.  of  inulin.  If  inulin  were  converted  in  the  body 
to  levulose,  then  a  large  increase  in  the  levulose  content 
of  the  urine  was  to  be  expected.  No  inulin  was  found  in 
t  he  feces,  but  the  patient  observed  a  strong  gas  formation 
in  the  intestine  during  the  period  following  the  meal, 
indicating  bacterial  decomposition  of  the  inulin.  B.  coli 
com  munis15  and  other  intestinal  bacteria  decompose 
inulin  without  any  production  of  sugar. 

No  enzymes  found  in  the  higher  animals  attack 
inulin,16  nor  do  yeast  invertin,17  malt-  or  "Taka"- 
diastase.18  That  plants  contain  an  inulase  has  been 
shown  by  Green,19  Bourquelot,20  Dean,21  and  others. 

Very  dilute  acids  (0.05  to  0.2  per  cent,  at  40  C, 
according  to  Chittenden18)  hydrolyze  inulin;  so  that  the 
possibility  of  an  inversion  by  the  gastric  juice,  and 
subsequent  utilization  must  be  taken  into  account. 
Eichaud22  has  shown  that  at  36  C.  0.1  per  cent,  hydro- 
chloric acid  inverts  86  per  cent,  of  inulin  in  twenty-four 
hours.  Bierry  and  Portier23  found  that  in  one  and  one- 
half  hours  at  38  C.  a  1  per  cent,  solution  of  inulin  was 
converted  to  levulose  by  a  gastric  juice  of  an  acidity 
equivalent  to  4.19  gm.  sodium  hvdroxid  per  liter.  In 
the  present  investigation  a  qualitative  experiment  to 
determine  whether  this  inverting  action  of  the  gastric 
juice  takes  place  in  vivo  was  performed.  Two  hundred 
c.c.  of  a  5  per  cent,  solution  of  inulin  were  introduced 
by  a  sound  into  the  stomach  of  a  dog  and  removed  after 
half  an  hour.  The  gastric  contents  contained  free  acid, 
but  gave  only  a  weak  reduction  with  Fehling's  solution. 
After  boiling  with  hydrochloric  acid  a  strong  reduction 
was  obtained,  indicating  that  the  inulin  had  not  left  the 

14.  Neubauer  :  Miinchen.  med.  Wchnschr.,  1905,  p.  1525. 

15.  Ducamp  :  Jahresb.  Thierchem.,  1907,  xxxvii,  952. 

16.  Swartz  :  Tr.  Connecticut  Acad.  Arts  and  Sc.,  1911,  xvi,  298  ;  a 
tabulation  of  the  literature  on  tbis  subject 

17.  Komanos  :  Jahresb.  Thierchem.,  1876,  vi,  180. 

18.  Chittenden  :  Am.  Jour.  Physiol,  1898,  ii.  17. 

19.  Green  :  Annals  of  Botany,  1888,  i,  223. 

20.  Bourquelot  :  Compt.  rend.,  1893,  cxvi,  1143. 

21.  Dean  :  Botanical  Gazette,  1903,  xxv,  69. 

22.  Richaud  :  Compt.  rend.  Soc.  de  biol.,  1900,  Hi,  416. 

23.  Bierry  and  Portier  :  Compt.  rend.  Soc.  de  biol.,  1900,  lii,  423. 


4 


stomach,  but  was  present  uninverted.  After  allowing 
the  gastric  contents  to  stand  two  hours  at  39  to  40  C,  a 
marked  reduction  was  obtained,  showing  that  the  acidity 
of  the  gastric  juice  was  sufficient  to  convert  inulin  into 
levulose  in  this  time. 

To  determine  whether  inulin  appears  in  the  feces, 
feeding  experiments  on  a  healthy  individual,  64.5  kg.  in 
weight,  were  tried.  In  the  first  experiment  the  diet  was 
not  weighed,  but  all  cellulose-containing  foods  were  care- 
fully avoided.  The  periods  were  divided-  as  follows : 
Fore,  two  clays;  experimental,  one  day  on  which  the 
inulin  was  taken  at  lunch  and  dinner;  after,  two  days. 
The  inulin  was  taken  chiefly  in  soup  or  mixed  with 
soft-boiled  eggs.  Although  comparatively  large  amounts 
(40  and  60  gm.)  were  fed,  the  subject  found  no  difficulty 


FEEDING  EXPERIMENTS 


Composition  of  Feces 
in  Periods 

Period 
(Days) 

Diet 

t  Moist 
) 

t  Air 
(Gm.) 

lydrates 
ililin 

lydrates 
Cent.) 

Series 

Weigh1 
(Gm. 

Weighi 
Dry 

Carbol 
as  Ir 
(Gm, 

o  Z 
U 

'\ 
•- 

Fore  =2 
Mid  =1 
After  =2 

fFore  =3 
Mid  =3 

.  After  =  1% 

Same  +  40  gm.  inulin. 
Same  as  fore-period .  . 

Same  +  60  gm.  inulin.* 
Same  as  fore-period .  . 

137 
182 
170 
215 
300 

52 
56 
50 
81 
89 
40 

1.62 

1.52 

0.8 

0.6 

1.31 

0.2 

3.1 
2.7 
1.6 
0.7 
1.4 
0.5 

*  Taken  in  one  meal  on  the  second  day  of  this  period. 


in  eating  the  inulin,  which  was  tasteless.  The  cellulose- 
free  diet  tended  toward  constipation,  so  that  in  the 
second  experiment  no  attempt  was  made  to  secure  a 
cellulose-free  diet,  but  rather  a  diet  approximately  con- 
stant in  its  cellulose  content.  The  periods  were  varied 
so  that  the  fore-  and  inulin  periods  each  were  of  three 
days'  duration.  In  the  inulin  period  a  day  before  and  a 
day  after  the  eating  of  the  inulin  were  included  to  insure 
against  any  of  the  carbohydrate  being  carried  into  the 
feces  of  the  after-period. 

Separation  of  the  feces  into  periods  was  accomplished 
with  charcoal  or  carmin  capsules.  The  feces  were 
weighed,  rubbed  to  a  paste  with  alcohol,  dried  on  a 
water-bath,  weighed  a  second  time,  and  ground.  From 


5 


the  well-mixed  sample  Prom  L.5  fco  4.5  gm.  were  heated 
on  a  water-bath  with  75  c.c.  water  and  7.5  c.c.  of  20  per 
cent,  hydrochloric  acid  for  from  twenty  to  thirty  minutes. 
The  gummy  materials  were  then  precipitated  with  phos- 
photungstic  acid,  filtered  off,  and  washed  with  hot  water. 
The  filtrate  was  neutralized,  evaporated  to  25  c.c, 
filtered  if  necessary,  and  a  sugar  determination  made  by 
Allihn's  gravimetric  method.  Check  determinations 
made  on  feces  to  which  a  known  weight  of  inulin  had 
been  added  before  drying  down  showed  that  by  the  above 
method  85  to  90  per  cent,  of  the  inulin  could  be 
recovered. 

No  increased  carbohydrate  content  of  the  feces  was 
observed  in  the  inulin  period  over  the  fore-  or  after- 
periods.  In  both  experiments  the  subject  noted  a  marked 
gas  formation  in  the  intestinal  tract.  This  began  in 
from  two  to  three  hours  after  the  meal  in  which  inulin 
was  eaten  and  continued  for  from  eight  to  ten  hours. 
The  gas  formation  was  in  harmony  with  the  observa- 
tions of  Neubauer.  In  the  experiments  recorded,  the 
tendency  of  the  subject  toward  constipation,  brought  on 
by  a  cellulose-free  diet,  doubtless  gave  more  opportunity 
for  the  action  of  the  bacteria  in  the  intestinal  tract.  In 
a  subject  without  this  tendency  to  constipation  larger 
amounts  of  inulin  might  well  be  recovered  in  the  feces. 

CONCLUSIONS 

1.  Inulin  fed  to  a  healthy  man  was  not  eliminated  in 
the  feces. 

2.  Marked  intestinal  fermentation  was  observed  to 
follow  the  feeding  of  inulin. 

3.  The  acidity  of  the  gastric  contents  of  a  dog  to 
which  inulin  was  given  by  a  stomach  sound  was  sufficient 
to  hydrolyze  inulin  partially  to  levulose  in  from  one  to 
two  hours. 

These  facts  would  seem  to  indicate  that  any  utiliza- 
tion of  inulin  can  occur  only  after  hydrolysis  by  the 
gastric  juice.  The  extent  of  this  hydrolysis  must  vary 
with  conditions  in  the  stomach.  If  the  diet  is  of  such  a 
character  that  it  leaves  the  stomach  soon,  the  action  of 
the  acid  gastric  juice  is  checked  by  the  intestinal  reaction 
before  the  inversion  of  inulin  can  proceed  far.  The 
acidity  of  the  gastric  contents  also  must  influence  the 
rate  of  inversion.  The  character  of  the  diet  and  individ- 
ual peculiarities  both  play  a  role  here.  Hence  the  per- 
centage utilization  of  inulin  for  any  individual  must 


6 


vary  and  cannot  be  determined  except  by  experiment. 
Any  inulin  which  leaves  the  stomach  unchanged  is  liable 
to  escape  utilization  and  undergo  bacterial  decomposition 
in  the  intestine,  a  decomposition  which  results  in  no 
formation  of  carbohydrates.  Any  inulin  which  escapes 
this  bacterial  action  is  probably  eliminated  unchanged 
in  the  feces. 

In  view  of  these  facts,  as  well  as  the  inability  to 
administer  more  than  comparatively  small  quantities, 
the  value  of  inulin  as  a  significant  source  of  energy  in 
human  dietaries  must  be  questioned. 

I  wish  to  acknowledge  my  indebtedness  to  Prof.  Lafayette 
B.  Mendel,  at  whose  suggestion  these  experiments  were  under- 
taken. 


Reprinted  from  The  Journal  of  the  American  Medical  Association 
April  20, 1912,  Vol.  LVIII,  pp.  1176  and  1177 

Copyright,  1912  , 
American  Medical  Association,  535  Dearborn  Ave.,  Chicago 


Reprinted  from  The  Journal  ok  BIOLOGICAL  Chemistry,  Vol.  XI,  No.  •>,  1912 


THE  INFLUENCE  OF  COCAINE  UPON  METABOLISM 
WITH  SPECIAL  REFERENCE  TO  THE  ELIM- 
INATION OF  LACTIC  ACID. 

By  FRANK  P.  UNDERHILL  and  CLARENCE  L.  BLACK. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 

New  Haven,  Connecticut.) 


(Received  for  publication,  February  27, 1912. ) 


The  introduction  of  cocaine  into  the  organism  is  followed  by  such 
well  defined  symptoms  that  an  almost  specific  influence  upon  the 
nervous  system  is  indicated.  In  the  main,  it  is  to  this  aspect  of 
its  action  upon  the  body  that  the  very  extensive  literature1  regard- 
ing this  drug  relates.  Definite  knowledge  of  the  effect  of  cocaine 
upon  general  metabolism  is  meagre  although  the  picture  presented 
by  the  cocaine  habitue*  is  sufficiently  characteristic  to  lead  one  to 
infer  that  ultimately  at  least  the  nutritional  rhythm  must  be  altered. 
The  widespread  employment  of  cocaine  as  an  ingredient  of  various 
types  of  proprietary  remedies  and  the  large  number  of  cases  of 
cocainism  makes  pertinent  at  this  time  an  inquiry  into  the  in- 
fluence upon  metabolism  of  the  drug  under  discussion. 

The  observation  of  Araki2  that  lactic  acid  appears  in  the  urine 
in  unusually  large  quantities  after  cocaine  injections  considered  in 
connection  with  the.findings  of  Wallace  and  Diamond3  that  cocaine 
causes  vacuolization  of  the  liver  cells  of  rabbits  suggested  the  pos- 
sibility of  a  disturbance  in  intermediary  metabolism.  In  the 
present  paper  the  relation  of  cocaine  poisoning  to  lactic  acid  out- 
put is  shown  and  the  influence  of  the  nutritive  condition  of  the 
animal  upon  this  type  of  acidosis  is  discussed.  It  is  also  demon- 
strated that  in  spite  of  the  marked  symptoms  characteristic  of 

1  Cf.  Richet:  Dictionnaire  de  physiologie,  iv,  p.  1,  1900. 

2  Araki:  Zeitschr.  f.  phyriol.  Chem.,  xv,  p.  335,  1891. 

3  Reported  at  the  19th  Annual  Meeting  of  the  American  Physiological 
Society,  New  York,  1907. 

255 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  XI,  NO.  3 


236        Influence  of  Cocaine  upon  Metabolism 

chronic  cocaine  poisoning  general  metabolism  is  only  slightly 
changed  from  the  normal  even  though  the  quantity  of  drug  admin- 
istered is  sufficient  to  finally  cause  death.  These  observations 
serve  as  a  further  illustration  of  the  tenacity  with  which  the  organ- 
ism adheres  to  the  fundamental  laws  underlying  its  metabolic 
processes;  in  other  words,  another  example  of  the  "factor  of  safety" 
principle  is  encountered  in  cocaine  poisorfing. 

THE  INFLUENCE  OF  COCAINE  UPON  METABOLISM,  AS  INDICATED 
BY  ITS  EFFECT  UPON  NITROGENOUS  EQUILIBRIUM  AND  PROTEIN 
AND  FAT  UTILIZATION. 

Methods.  The  experiments  were  planned  so  that  the  animals 
(dog  and  rabbit)  employed  were  kept  upon  a  fixed  diet  and  cocaine 
administered  subcutaneously  at  a  time  sufficiently  long  after  a 
meal  to  avoid  the  danger  of  food  being  vomited.  During  the  first 
period  of  the  experiments  the  drug  was  given  once  daily,  later 
the  animal  was  kept  under  the  influence  of  cocaine  the  greater 
portion  of  each  day  by  repetition  of  the  injection. 

Lactic  acid  was  estimated  by  the  Ryffel4  procedure.  The 
Folin  method  as  modified  by  Steel5  was  employed  in  the  deter- 
mination of  ammonia  in  the  urine  of  rabbits.  The  other  deter- 
minations were  carried  out  according  to  the  well  known  meth- 
ods usually  employed  in  this  laboratory.  Urine  was  collected  in 
twenty-four  hour  periods  by  catheterization  (dogs)  or  by  pressure 
on  the  bladder  through  the  body  wall  (rabbits) .  Unless  otherwise 
noted  all  urines  of  dogs  were  acid  in  reaction  to  litmus.  The  rab- 
bits' urines  were  alkaline  throughout. 

Description  of  experiments.  Experiments  1  and  2.  In  these 
observations  dogs  50  and  51  were  kept  for  several  days  previous  to 
the  investigation  upon  the  diet  arranged  for  the  experimental 
trials  in  order  to  bring  them  as  nearly  as  possible  into  a  condition 
of  nitrogenous  equilibrium.  A  fore-period  was  followed  by  an 
interval  during  which  the  animals  received  daily  subcutaneous 
injections  of  cocaine  hydrochloride  (Kahlbaum's  crystalline  pro- 
duct) dissolved  in  water.  In  addition  to  a  constant  diet  through- 
out the  experiment  the  animals  received,  also,  a  fixed  water  intake. 

4  Ryffel:  Journ.  of  Physiol.,  xxxix,  p.  v.  1909-10. 

5  Steel:  This  Journal,  viii,  p.  365,  1910-11. 


Frank  P.  Underhill  and  Clarence  L.  Black  237 


Protocol  of  Experiment  1. 

Dog  50,  weighing  12.8  kilos,  was  normal  in  every  respect  except  that  she 
was  extremely  deaf.  The  diet  consisted  of  200  grams  meat,  80  grams  cracker 
meal,  40  grams  lard,  10  grams  bone  ash,  and  300  cc.  water.  The  total  nitro- 
gen intake  amounted  to  7.40  grams  nitrogen  daily  with  sufficient  fat  and 
carbohydrate  to  yield  approximately  70  calories  fuel  value  per  kilo  of  body 
weight.  Each  day  food  was  given  at  9 :30  a.m.  and  the  first  cocaine  injection 
at  3:30  p.m. 

On  October  20  the  cocaine  period  was  begun.  Just  before  the  cocaine 
injection  the  rectal  temperature  was  38.6°  C.  and  two  hours  later  had  risen 
to  39.0°  C.    The  pupils  showed  extreme  dilatation. 

October  21.  In  the  morning  the  dog  seemed  normal  and  ate  food  with 
evident  relish.  Temperature  before  cocaine  administration  was  38.6°  C.  and 
had  risen  to  40.0°  C.  two  hours  later.  About  45  minutes  after  the  injection  the 
animal  exhibited  peculiar  movements  of  the  head  which  were  constant. 
The  dog  was  extremely  restless.    The  pupils  were  greatly  dilated. 

October  22.  The  dog  was  apparently  normal  at  meal  time.  Symptoms 
after  cocaine  injection  similar  to  those  of  previous  days. 

October  22.    Symptoms  unchanged. 

October  24.  Rectal  temperature  at  9:30  a.m.  =  38.8°  C,  just  before 
injection  at  3:30  p.m.  =  38.6°  C. ;  at  4:30  p.m.  =  40.9°  C. 

At  4:30  p.m.  the  heart  action  was  very  slow  but  strong.  Arhythmic 
beating  was  in  evidence.  There  was  extreme  dilatation  of  pupil.  The 
animal  was  very  much  excited  and  the  head  was  constantly  moved  up  and 
down.  Usually  the  animal  was  too  deaf  to  pay  attention  to  any  sound,  but 
at  this  time  it  would  respond  to  a  call. 

October"25.  In  the  morning  the  dog  appeared  normal  and  devoured  food 
as  usual. 

Temperature  at  9:30  a.m.  =  38.8°  C,  just  before  injection;  at  3:30  p.m. 
=  38.8°  C;  at  5:00  p.m.  =  41.1°  C. 

The  movements  of  animal  were  more  pronounced  and  there  was  much 
more  excitation  after  cocaine  administration  than  had  been  observed  at  any 
previous  time.  The  peculiar  irregularity  of  the  heart  was  again  in  evidence 
at  5:00  p.m.  although  previous  to  the  injection,  the  beat  was  normal. 

October  26.    The  appetite  of  animal  was  ravenous. 

Temperature  at  9:30  a.m.  =  38.6°  C,  just  before  injection;  at  3:30  p.m. 
=  38.6°  C,  at  4:00  p.m.  =  41.6°  C  at  5:00  p.m.  =  40.9°  C. 

It  was  apparent  that  the  animal  had  become  much  more  sensitive  to  the 
cocaine  since  the  usual  daily  injection  was  followed  by  greatly  augmented 
symptoms  of  excitation.    These  lasted  for  a  period  of  two  hours. 

October  27.    The  dog  devoured  food  with  apparent  relish. 

Temperature  at  3:15,  just  before  injection  =  38.6°  C;  at  3:45  =  41.2°  C. ; 
at  4:15  =  41.6°  C;  at  4:45  =  409°  C. ;  at  5:15  =  39.8°  C. 

The  symptoms  of  excitation  and  pupil  dilatation  appeared  within  fif- 
teen minutes  after  cocaine  administration.  Apparently  the  peculiar  head 
movements  were  caused  by  an  attempt  to  push  the  head  out  of  the  cage 


238        Influence  of  Cocaine  upon  Metabolism 


TABLE  1. 
Experiment  I — Dog  50. 
Fore  Period. 


(Daily  Nitrogen  Intake  =  7.40  grams.) 


H 

URINE 

FECES 

O 
0 

fa 

DATE  Z 

H 
M 

vity 

c 

<D 
M 

itrogen 

Weight 

a 

1 

a 

DD 
O 

0 

u 
O 

O 

z; 

c3 

O 

s 

u 

A.ILT  D 

I 

- 

0 

0 

olume 

pecific 

otal  N 

mmon: 

actic  A 

to 

0 

>» 

u 

ater 

otal  Ni 

ther  E: 

05 

> 

w 

H 

< 

Q 

'  Eh 

H 

1910  mgms. 

kilos 

cc. 

gms. 

gms. 



mgms. 

gms. 

gms. 

per 
cent 

gms. 

gms. 

October 

15 

12.8 

300 

1.020 

6.15 

0.28 

49 

26.8  11.6 

56 

*(4.5) 

16 

12.8 

300 

1.020 

6.18 

0.30 

49 

32.6 

14.0 

57 

(4.8) 

17 

12.8 

310 

1.021 

6.03 

0.27 

48 

19.0 

12.5 

34 

3.30 

6.09 

(4.4) 

-18 

J2.8 

300 

1.020 

6.24 

0.29 

49 

29.0 

15.0 

49 

(4.6) 

19 

12.8 

300 

1.020 

6.18 

0.26 

48 

96.0 

46.0 

51 

(4.2) 

Average 

per  day.. .  . 

12.8 

302 

1.020 

6.15 

0.28 

48 

40.7 

19.8 

49 

0.66 

1.22 

(4.5) 

J^irsi  Cocaine  Period. 


20 

128 

12 

8 

410 

1 

.020 

6 

45 

0.26 
(4.3) 

54 

51 

0 

26 

49 

21 

128 

12 

7 

275 

1 

.025 

5 

88 

0.24 
(4.0) 

56 

60 

0 

28 

0 

53 

.  22 

128 

12 

6  260 

1 

020 

6 

15 

0.34 

60 

29 

0 

15 

0. 

49 

(5.5) 

23 

128 

12 

6  270 

1 

021 

5 

64 

.28 

64 

35 

0 

17 

0 

51 

(4.9) 

1 

24 

128 

12 

6  210 

1 

026 

5 

73 

0.35 

61 

47 

0 

24 

0 

49 

(6.1) 

25 

128 

12 

5 

240 

1 

030 

6 

75 

0.34 
(5.0) 

54 

35 

0 

20 

0 

42 

8.0717.83 

26 

128 

12 

4 

170 

1 

040 

6. 

66 

0.30 
(4.5) 

53 

51 

0 

30 

0 

41 

*  Figures  in  brackets  indicate  percentages  of  total  nitrogen. 


Frank  P.  Underhill  and  Clarence  L.  Black  239 

TABLE  1— Continued 

First  Cocaine  Period — Continued 


tngms 

128 
128 
128 
128 


128 


kilos 

12.3 
12.3 
12.2 
12.2 


165  I  1.045 
160  I  1.046 
170  j  1.040 
165  1.041 


12.5"  227  i  1.032 


gms. 


gnts. 


12  0.27 
(4.4) 
6.54|  0.30 
■  (4.5) 
6.12  0.33 
' (5.3) 
6.06!  0.30 
4.9) 


6.19|  0.30 

I  (4.8) 


Weight 


70 
76 
71 

78 


63 


cent  sms- 


40.0  24  .0  40 

50.0  24.0  52 

47.0  25.0  47 

44.0,24.0  45 


41.0  23.0  47 


0.73  1.62 


Second  Cocaine  Period. 


November 

1 

256 

12.2 

170 

1.040 

8.25 

0.36 
(4.3) 

84 

46.0 

21.0 

54 

2 

256 

11.4 

140 

1.050 

6.84 

0.35 
(5.1) 

83 

52.0 

32.0 

39 

3 

256 

11.3 

120 

1.052 

5.94 

0.31 
(5.2) 

79 

53.0 

33.0 

38 

3.55 

13.95 

4 

256 

11.2 

125 

1.050 

6.18 

0.31 
(5.0) 

80 

50.0 

23.0 

54 

Average 

per  day.  .  . 

256 

11.5 

138 

1.048 

6.80 

0.33 

(4.9) 

81 

50.0 

27.0 

46 

0.88 

3.48 

240        Influence  of  Cocaine  upon  Metabolism 


Balances 


Fore  Period 

grams 

Nitrogen  in  food  37.00 

Nitrogen  in  excreta: 

Urine   30.78 

Feces   3.30  34.08 


Nitrogen  balance   +2.92 

Per  day   +0.58 

Nitrogen  Utilization  =  91  percent. 


Ether  extract  in  food . 
Ether  extract  in  feces . 


grams 

323.20 
.  6.09 


Fat  utilized. . . 
Fat  utilization 


. ..  317.11 
per  cent. 


First  Cocaine  Period. 


Nitrogen  in  food   81.40 

Nitrogen  in  excreta : 

Urine  68.10 

Feces   8.07  76.17 


Ether  extract  in  food   711.04 

Ether  extract  in  feces   17.83 


Nitrogen  balance   +5.23 

Per  day   +0.47 

Nitrogen  utilization  =  90  per  cent. 


Fat  utilized   693.21 

Fat  utilization  =  98  per  cent. 


Second  Cocaine  Period. 


grams 

Nitrogen  in  food   29  .60 

Nitrogen  in  excreta: 

Urine   27.21 

Feces   3.55  30.76 


Ether  extract  in  food  258 . 56 

Ether  extract  in  feces   13  .95 


Nitrogen  balance  —  1.16 

Per  day  -  0.29 

Nitrogen  utilization  =  88  per  cent. 


Fat  utilized  244  .61 

Fat  utilization  =  94  per  cent. 


toward  the  light.  During  the  remainder  of  this  period  which  was  concluded 
on  October  31  no  new  features  developed. 

It  was  planned  to  begin  the  second  cocaine  period  on  October  31  by  giving 
two  injections  of  the  drug,  at  12:00  m.  and  4 :00  p.m.  respectively.  The  first 
injection  caused  vomiting  which  contaminated  the  urine.  This  period  was 
therefore,  commenced  on  the  next  day,  November  1.  On  this  date  cocaine 
in  doses  of  128  mgrms.  each  was  administered  at  3:00  p.m.  and  5:00  p.m. 
Just  previous  to  the  first  injection  the  temperature  was  38.5°  C,  at  5:p.m.; 
40.0°  C,  at  6:00,  p.m.,  40.9°  C.  The  dog  was  in  a  state  of  extreme  activity 
during  this  time. 

November  2.  Cocaine  was  injected  as  on  November  1.  The  conditions 
of  the  animal  had,  however,  undergone  a  marked  change  since  all  movements 
were  executed  in  a  weak  and  uncertain  manner. 


Frank  P.  Under  hi  11  and  Clarence  L.  Black  241 

TABLE  2. 
Dog  51. 
Fore  Period. 


(Daily  Intake  of  Nitrogen  =  4.72  grams) 


w 

URINE 

FECES 

< 

0 
0 
0 

a 

4> 

U) 

Weight 

O 

DATE 

DAILY  DOSE  OF 

BODY  WEIGHT 

B 

"0 
> 

Specific  Gravit; 

Total  Nitrogen 

Ammonia  Nitr 

Lactic  Acid 

0 

>> 

u 

Q 

u 

1 

Total  Nitrogen 

Ether  Extract 

1910 

mgms. 

kilos 

cc. 

firms. 

gms. 

mgms. 

per 
cent 

gms. 

gms. 

November 

30 

8.3 

120 

1.040 

4.56 

0.21 

*(4.6) 

45 

December 

1 

8.2 

175 

1.035 

4.25 

0.23 
(5.1) 

46 

52.0 

23.0 

56 

2 

8.2 

165 

1.036 

4.23 

0.23 
(5.1) 

51 

17.0 

10.0 

42 

3 

8.2 

165 

1.036 

4.24 

0.17 
(4.0) 

51 

29.0 

18.0 

38 

2.10 

5.46 

4 

8.2 

125 

1.040 

4.21 

0.16 

(3.7) 

45 

20.0 

11.0 

45 

5 

8.2 

175 

1.030 

4.20 

0.18 

(4.2) 

48 

15.0 

10.0 

33 

Average 

per  day. 

8.2 

154 

1.036 

4.28 

0.19 

(4.4) 

47 

22.0 

12.0 

42 

0.35 

0.91 

Cocaine  Period. 

6  123  8.2 

7  123  j  7.6 

8  123  j  7.6 

9  123  7.6 
10      123  j  7.6 


220 
200 
155 
150 


1.026 
1.030 
1.035 
1.034 


4.44    0.18|    58     11.0  5.0  55 

(4.0); 

3.84    0.14'    73    23.0  11.0  51 

(3.6) 
4.35    0.16  74 

I  (3.6)1 
4.29  0.17 


70 


(3.9), 


155   1.035  3.80    0.16|    79     42.0  22.0  46 

(4.2) 


*  Figures  in  brackets  indicate  percentages  of  total  nitrogen. 


242        Influence  of  Cocaine  upon  Metabolism 


TABLE  2— Continued 

Cocaine  Period — Continued 


1910 

December 
11 

12 

13 

14 

15 


mgms.\  kilos 


123 
123 
123 
123 
123 


Average 
per  day.  123 


7.6 
7.6 


250 
130 


7.5  140 


7.5 
7.5 


7.6 


135 
145 


o  s 


i  2 


m     ,  EH 


I  gms.  gms. 

1.030  4.21  0.18 
(4.2) 

1.040  4.50  0.18 
(4.0) 
0.16 

(5-5) 


1.035  2.88 

1.037  3.15 

j 

1.033  4.56 


Weight 


gms. 


168 


0.16 
(5.0) 
0.19 
(4.1) 


1.033  4.00 


0.17 

(4.2) 


85 


90   j 50.0 

79   1 11.0 

90   j 44.0 
j  20  .0 
79  49.0 


per 
cent 


78  :25.0 


25.0,  50 


6.0 


H    j  W 
gms.  j  gms. 

3.89  (32.54 


45 


31.0!  30 
12.0  40 


25.0 


49 


13.7   45  0.39!  3.25 


Balances 
Fore  Period. 

grams  grams 

Nitrogen  in  food   28.32     Ether  extract  in  food   243.60 


Nitrogen  in  excreta: 

Urine   25.69 

Feces   2.10 


Ether  extract  in  feces   5.46 

Fat  utilized   238.14 

Fat  utilization  =  97  pei  cent. 


Nitrogen  balance   +0.53 

Per  day   +0.08 

Nitrogen  utilization  =  92  per  cent. 

Cocaine  Period. 

grams  grams 

Nitrogen  in  food   47.20     Ether  extract  in  food  406.00 


Nitrogen  in  excreta : 

Urine  40.02 

Feces   3.89  43.91 

Nitrogen  balance   +3.29 

Per  day   +0.33 

Nitrogen  utilization  =  91  per  cent. 


Ether  extract  in  feces . 


32.54 


Fat  utilized   373.46 

Fat  utilization  =  91  per  cent. 


Frank  P.  Underhill  and  Clarence  L.  Black  243 


November  8.  The  dog  showed  signs  of  diminished  appetite.  Conditions 
remained  unchanged. 

November  4-    Conditions  about  as  usual.    Animal  appears  weak. 

November  5.  The  dog  died  twenty-five  minutes  after  the  first  cocaine 
injection.  Just  before  death  the  dog  was  in  a  state  of  extreme  activity. 
This  was  rapidly  followed  by  a  period  of  partial  paralysis  culminating  in 
respiratory  failure.  Further  data  concerning  this  experiment  may  be  found 
in  Table  1,  pp.  238-240. 

Protocol  of  Experiment  2.    Dog  51 . 

A  fox  terrier  bitch  of  8.3  kilos  was  placed  upon  a  fixed  diet  composed  of 
125  grams  meat,  60  grams  cracker  meal,  20  grams  lard,  10  grams  bone  ash 
and  150  cc.  water  for  a  period  of  10  days  previous  to  the  actual  fore  period 
of  the  experiment.  The  nitrogen  content  of  this  diet  amounted  to  4.72 
grams;  the  fuel  value  was  approximately  69  calories  per  kilo  body  weight. 

November  30.    On  this  date  the  fore  period  of  six  days  was  begun. 

December  6.  The  cocaine  period  was  commenced  by  the  injection  of  123 
mgms.  cocaine  at  3:00  p.m.  No  rise  in  temperature  could  be  observed. 
The  only  symptoms  noticeable  were  salivation  and  pupil  dilatation. 

December  7.  About  one-half  hour  after  the  administration  of  cocaine 
the  dog  became  markedly  excited,  the  bodily  movements  not  being  under 
perfect  control.  Pupil  dilatation  was  extreme  and  the  arhythmic  heart 
beat  was  evident. 

Each  day  up  to  December  12  the  symptoms  of  excitement  etc.  were 
noticeable  but  unchanged  in  character. 

December  12.  Shortly  after  the  cocaine  injection  the  animal  became 
completely  paralyzed  in  the  hind-quarters.  The  j  aws  and  tongue  were  kept 
constantly  in  motion  as  though  the  animal  was  tasting  something  unpleas- 
ant. The  dog  remained  in  this  condition  for  several  hours  during  which 
she  appeared  deaf  and  blind. 

December  13.  The  animal  seemed  normal  although  somewhat  Weak. 
The  weakness  became  more  and  more  noticeable  and  on  December  15  the 
experiment  was  terminated. 

For  other  data  associated  with  this  animal  see  Table  2,  pp.  241-242. 

DISCUSSION  OF  RESULTS. 

From  the  details  of  the  protocols  and  tables  submitted  it  is 
apparent  that  the  most  obvious  symptoms  arising  from  cocaine 
injections  in  the  doses  given  are  distinctly  of  nervous  origin.  A 
significant  influence  is  also  exerted  upon  the  heat  regulating  mech- 
anism whereby  the  temperature  is  quite  markedly  increased  for 
a  short  period  after  which  there  is  a  gradual  return  to  the  nor- 
mal.6   With  daily  doses  of  10  mgms.  of  cocaine  hydrochloride 

•Reichert:  Centralbl.  f.  d.  med.  Wissenschaften,  1889,  p.  444. 


244        Influence  of  Cocaine  upon  Metabolism 

per  kilo  of  body  weight  no  appreciable  influence  can  be  detected 
upon  the  course  of  nitrogenous  metabolism  nor  upon  the  utiliza- 
tion of  protein  and  fat  although  body  weight  shows  an  appreciable 
decline. 

When  injections  of  15  mgms.  cocaine  per  kilo  are  daily  admin- 
istered fat  utilization  is  very  slightly  impaired  and  is  accompanied 
by  a  decreased  body  weight.  Doses  of  20  mgms.  per  kilo  per  day 
divided  into  two  injections  show  a  fairly  distinct  detrimental  in- 
fluence upon  both  protein  and  fat  utilization  and  for  the  first  time 
a  slight  negative  balance  was  in  order.  Body  weight  was  markedly 
diminished  under  this  dosage. 

The  water  excretion  of  Dog.  50  was  quite  distinctly  diminished 
under  cocaine  when  compared  with  that  of  the  fore-period.  This 
finding  does  not  hold  true  for  Dog  51.  The  difference  may  be 
explained  perhaps  by  the  fact  that  Dog  50  was  apparently  much 
more  sensitive  in  its  reaction  to  cocaine  with  respect  to  the  tem- 
perature raising  influence  than  was  Dog  51.  Assuming  this  to  be 
true  more  water  was  probably  eliminated  by  the  lungs  in  the  first 
case  than  in  the  second  which  would  account  for  lessened  water 
elimination  by  the  kidney. 

THE  INFLUENCE  OF  COCAINE  UPON  THE  ELIMINATION  OF  LACTIC 
ACID  IN  THE  URINE. 

The  presence  of  lactic  acid  in  the  urine  in  appreciable  quanti- 
ties has  been  a  subject  of  much  investigation  and  discussion  result- 
ing in  a  multiplicity  of  conflicting  theories  with  respect  to  its  sig- 
nificance. Out  of  the  enormous  literature7  relative  to  lactic  acid 
only  a  few  references  that  have  a  bearing  upon  the  present  paper 
may  be  cited. 

Thus,  Araki8  has  demonstrated  that  lactic  acid  appears  in  the 
urine  in  the  absence  of  a  sufficient  supply  of  oxygen  induced  by 
various  types  of  toxic  compounds  and  epileptic  seizures.  The  older 
work  of  Spiro9  indicating  that  increased  muscular  activity  leads 
to  lactic  acid  excretion  finds  confirmation  in  the  recent  investiga- 

7  Ryffel:  Quarterly  Journ.  of  Med.,  in,  p.  413,  1909-10. 

8  Araki:  loc.  cit. 

9  Spiro:  Zeitschr.  f.  physiol.  Chem.,  i,  p.  Ill,  1877. 


Frank  P.  Underhill  and  Clarence  L.  Black  245 


tions  of  Ryffcl10  and  Feldman  and  Hill.11  According  to  the  latter 
authors  the  appearance  of  lactic  acid  in  the  urine  may  be  greatly 
diminished  by  breathing  oxygen  before  and  after  exertion.  They 
conclude  that  the  increased  production  of  lactic  acid  by  the  muscles 
is  due  to  oxygen  want,  a  view  that  was  earlier  denied  by  Ryffel.12 

Viewed  from  the  standpoint  of  ultimate  origin,  it  is  possible  that 
lactic  acid  is  intimately  associated  with  the  carbohydrate  store  of 
the  body;  for  Araki  found,  under  the  experimental  conditions,  less 
lactic  acid  in  the  urine  of  starving  animals  than  could  be  dem- 
onstrated in  the  urine  of  those  well  fed.  On  the  other  hand, 
phosphorus,  which  leads  to  a  disappearance  of  the  carbohydrate 
store,  causes  a  large  output  of  lactic  acid  which  may  be  accompan- 
ied by  an  increased  elimination  of  ammonia.13  It  is  presumed  that 
the  increase  of  the  latter  urinary  constituent  is  for  the  purpose  of 
neutralizing  the  lactic  acid  produced. 

In  the  experiments  to  be  recorded  the  rabbits  were  kept  upon  a 
diet  consisting  of  300  grams  of  carrots  and  20  grams  oats  which 
experience  had  demonstrated  would  usually  be  entirely  eaten  each 
day. 

Experiment  3.    Rabbit  B. 

During  each  day  of  the  fore  period  this  animal  left  small  portions  of  the 
carrots  uneaten.  After  the  subcutaneous  cocaine  injections  no  food  was 
ever  left.  For  the  first  two  days  of  the  cocaine  period  no  evidences  of  ab- 
normal symptoms  were  observed.  On  the  third  day,  however,  there  was 
considerable  dilatation  of  the  pupil.  Beginning  with  November  9,  the  tenth 
day  of  administration,  irritability  and  restlessness  were  noticeable.  The 
appetite  remained  good,  all  food  being  eaten  shortly  after  the  daily  cocaine 
administration.  About  10  minutes  after  cocaine  injection  on  November  11 
the  animal  was  seized  with  convulsions  and  respiration  almost  ceased,  but 
recovery  was  complete  three-quarters  of  an  hour  later.  On  the  succeeding 
two  days  convulsions  were  in  evidence  shortly  after  cocaine  administration, 
but  in  each  instance  recovery  was  complete.  The  animal  died  in  a  convul- 
sion on  November  14.  The  liver  which  was  immediately  excised  contained 
8  per  cent  of  glycogen. 

From  the  data  in  Table  3  it  will  be  observed  that  the  injections  of  cocaine 
were  progressively  increased  from  approximately  15  mgms.  per  kilo  to  20 


10  Ryffel:  Journ.  of  Physiol.,  xxxix,  p.  xxix,  1909. 

11  Feldman  and  Hill:  Journ.  of  Physiol.,  xlii.  p.  439.  1911. 

12  Ryffel:  Journ.  of  Physiol.,  xxxix,  p.  xxix,  1909. 

13  Mandel  and  Lusk:  Amer.  Journ.  of  Physiol.,  xvi,  p.  129,  1906. 


246       Influence  of  Cocaine  upon  Metabolism 

TABLE  3. 
Rabbit  B. 


Fore  Period. 


DATE 

DAILY 
DOSE  OF 
COCAINE 

BODY 
WEIGHT 

URINE 

Volume 

Specific  1 
Giavity 

Total 
Nitrogen 

Ammonia 
Nitrogen 

Lactic 
Acid 

1910 

October 
26 

27 

28 

zy 

30 

mgms. 

kilos 

2.38 
2.38 
2.34 
2.32 
2.32 

cc. 

200 
110 
120 
105 
125 

1.016 
1.025 
1.024 
1.025 
1.025 

grams 

1.00 
0.76 
0.75 
0.93 
0.96 

mgms. 

2.5 
(0.25)* 

1.8 
(0.23) 

1.8 

(0.23) 

1  0 
1 . 8 

(CI  1  q\ 

(u. iy; 

1  A 

1 .4 
(r\  1 

(0.15) 

mgms. 

9 
8 
10 

1  A 
1  O 

J  z 

Average 
v  per  day . . 

2.34 

132 

1.023 

0.88 

1.8 
(0.21) 

10 

Cocaine  Period. 

31 

33 

2.32 

125 

1.025 

0.90 

1.0 

11 

(0.11) 

November 

1 

34.5 

2.32 

160 

1.021 

0.97 

1.08 

11 

(0.19) 

2 

34.5 

2.32 

190 

1.020 

0.93 

2.7 

13 

(0.29) 

3 

34.5 

2.32 

215 

1.018 

0.80 

2.7 

10 

(0.33) 

4 

34.5 

2.32 

215 

1.019 

0.74 

2.3 

13 

(0.31) 

5 

34.5 

2.32 

250 

1.015 

0.71 

2.7 

12 

(0.18) 

6 

34.5 

2.32 

210 

1.016 

0.73 

1.8 

14 

(0.24) 

7 

46 

2.30 

185 

1.018 

0.67 

1.8 

12 

(0.27) 

8 

57.6 

2.30 

210 

1.016 

0.59 

1.8 

15 

(0.30) 

Frank  P.  Underhill  and  Clarence  L.  Black  247 


TABLE  3  Continued 
( loca  inc.  Period — Continued 


DATE 

DAILY 
DOSE  OF 
COCAINE 

BODY 
WEIGHT 

1910 

7TXQ  TTl  S 

November 

9 

69 

2.26 

10 

89 

2.22 

11 

101 

2.24 

12 

101 

2.28 

13 

101 

2.30 

Average 
per  day  .  . 

2.29 

Volume 


235 
195 
180 
200 
185 

196 


0  pGCtnc 

Total 

A  m  mon \n 

JU9;Ct'lC 

Gravity 

N  ltrogen 

Nitrogen 

Acid 

grams 

mgms. 

mgms. 

1.015 

0.61 

1.8 

26 

(0.30) 

1.020 

0.61 

6.3 

25 

(1.0) 

1.020 

0.63 

1.8 

33 

(0.28) 

1.024 

0.82 

1.1 

39 

(0.13) 

1.024 

0.88 

1.1 

51 

(0.12) 

1.018 

0.75 

2.2 

20 

(0.30) 

*  Figures  in  brackets  indicate  percentages  of  total  nitrogen. 

mgms.  on  November  7,  to  25  mgms.  per  kilo  on  November  8,  to  30  mgms. 
on  November  9,  to  40  mgms.  on  November  10,  and  finally  to  45  mgms.  per 
kilo  on  November  11.  Frequent  tests  throughout  the  cocaine  period  failed 
to  demonstrate  an  appreciable  rise  in  rectal  temperature. 

Experiment  4-    Rabbit  C. 

This  animal  behaved  in  a  manner  very  similar  to  Rabbit  B.  A  rise  in 
rectal  temperature  of  about  0.5°  C.  was  the  maximum  increa  e  shown  dur- 
ing the  period  of  observation.  The  daily  dose  of  cocaine  given  varied  from 
approximately  10  mgms.  per  kilo  on  November  29  and  30,  to  20  mgms.  on 
December  1  to  6  inclusive,  and  from  this  time  to  the  end  of  the  experiment 
the  animal  received  approximately  34  mgms.  cocaine  per  kilo  body  weight. 

From  the  data  in  Tables  3  and  4  with  rabbits  and  those  in 
Tables  1  and  2  with  dogs,  it  is  evident  that  cocaine  causes  an 
appreciable  increase  in  the  elimination  of  lactic  acid  in  the  urine. 
In  a  general  way  the  quantity  of  lactic  acid  thus  excreted  is  in 
direct  proportion  to  the  amount  of  cocaine  injected.  The  output 
of  ammonia,  however,  does  not  appear  to  be  significantly  increased 
by  the  augmented  elimination  of  lactic  acid,  an  indication  that  in 


248        Influence  of  Cocaine  upon  Metabolism 

TABLE  4. 

Rabbit  C. 


Fore  Period. 


DATE 

DAILY 

BODY 

URINE 

DOSE  OF 
COCAINE 

WEIGHT 

Volume 

Specific 
Gravity 

Total 
Nitrogen 

Ammonia 
Nitrogen 

Lactic 
Acid 

mgms. 

kilos . 

c.c. 

grams 

mgms. 

mgms. 

1910 

November 

21 

2.26 

290 

1.014 

0.80 

1.0 

24 

22 

2.24 

295 

1.014 

0.85 

1.0 
CO  12) 

20 

23 

2.20 

245 

1.015 

0.83 

3.6 
(0.43) 

23 

24 

2.18 

235 

1.016 

0.83 

4.5 
(0.54) 

22 

25 

2.20 

230 

1.016 

0.80 

3.6 
(0.45) 

22 

26 

2.22 

190 

1.018 

0.82 

3.6 
(0.44) 

20 

27 

2.24 

200 

1.019 

0.81 

4.5 
(0.55) 

21 

.  28 

2.26 

230 

1.018 

0.83 

4.5 
(0.54) 

23 

Average 

per  day  .  . 

2.22 

239 

1.016 

0.82 

3.2 
(0.39) 

22 

Cocaine  Period. 

29 

20 

2.26 

270 

1.017 

1.18 

4.5 
(0.38) 

23 

30 

20 

2.24 

245 

1.020 

1.40 

4.5 
(0.32) 

23 

December 

1 

45 

2.26 

240 

1.021 

1.30 

3.6 
(0.27) 

24 

2 

45 

2.24 

230 

1.021 

1.26 

3.6 
(0.27) 

24 

3 

45 

2.26 

220 

1.022 

0.97 

3.6 
(0.37) 

25 

4 

45 

2.30 

230 

1.022 

0.86 

4.5 

(0.52) 

26 

Frank  P.  Underhill  and  Clarence  L.  Black  249 


TABLE  4— Continued. 


DATE 

DAILY 

BODY 

UKINK 

DOSE  OP 
COCAINE 

WEIGHT 

Specific 

Total 

Ammonia 

Lactic 

Volume 

(  tT*M  Vi  t  V 

V_X J      V  H/J 

Nitrogen 

Nitrogen 

Acid 

mgms. 



kilos. 

c.c. 

grams 

mgms. 

mgms. 

December 

0 

O  QA 
Z .  oU 

91  ^ 

1  099 
1  .  \}ZL 

0 

^0 

OU 

/lo^ 

a 

0 

O  Qfl 

Z .  oU 

215 

1  099 

0  SO 

00 

(0.67) 

7 

75 

O  OA 

205 

1.022 

0.74 

6.3 

33 

(0.85) 

8 

75 

2.24 

225 

1.020 

0.72 

8.1 

36 

• 

(1.12) 

9 

75 

2.26 

190 

1.024 

0.83 

9.0 

40 

(1.08) 

10 

75 

2.30 

230 

1.020 

0.95 

9.0 

42 

• 

(0.94) 

Average 

2.26 

226 

1.022 

0.99 

5.5 

30 

per  day  . 

(0.55) 

*  Figures  in  brackets  indicate  percentages  of  total  nitiogen. 


this  connection  lactic  acid  may  be  neutralized  by  some  base  other 
than  ammonia.  This  is  particularly  true  for  dogs,  but  does  not 
hold  quite  so  well  with  rabbits,  for  with  Rabbit  C.  the  output  of 
ammonia  paralleled  closely  the  elimination  of  lactic  acid. 

The  influence  of  diet  upon  lactic  acid  elimination  under  the 
experimental  conditions  may  be  indirectly  inferred  from  the  data 
of  .  Table  5  obtained  from  Dog  52  during  a  period  of  inanition. 
Here  it  will  be  observed  that  in  spite  of  largely  increased  doses  of 
cocaine  lactic  acid  output  fell  considerably.  The  larger  quantities 
of  lactic  acid  excreted  during  the  first  few  days  of  the  experiment 
may  perhaps  be  explained  on  the  assumption  that  the  carbohy- 
drate store  of  the  body  during  this  interval  had  not  been  depleted. 
As  soon  as  this  condition  had  been  reached  a  diminution  in  lactic 
acid  output  took  place.  These  results  are  in  harmony  with  the 
theories  outlined  by  Araki,  but  are  in  opposition  to  the  observa- 
tions reported  for  pernicious  vomiting  of  pregnancy  where  lactic 
acid  is  eliminated14  in  the  urine  probably  as  a  result  of  the  inanition 

"Underhill:  This  Journal,  ii,  p.  485,  1906-07;  see  also  Underhill  and 
Rand:  Arch,  of  Int.  Med.,  v,  p.  61,  1911. 


250 


Influence  of  Cocaine  upon  Metabolism 


TABLE  5. 
Dog  52 — Inanition. 


DAILY 

URINE 

DATE 

DOSE  OF 
.  COCAINE 

BODY 

WEIGHT 

Volume 

Specific 

Total 

Ammonia 

Lactic 

Gravity 

Nitrogen 

Nitrogen 

Acid 

rngms. 

kilos 

cc. 



grams 

gram. 

mgms. 

1910 

■ 

November 

10 

120 

10.2 

160 

1.050 

6.57 

0.31 

(4.7)* 

41 

11 

120 

10.2 

120 

1.058 

4.23 

0.39 
(9.2) 

38 

12 

120 

9.9 

180 

1.025 

3.06 

0.34 
(11.1) 

39 

13 

120 

9.6 

140 

1.035 

2.73 

0.26 

(9.5) 

36 

14 

120 

9.3 

|  200 

15 

150 
120 

9.0 

1.040 

4.92 

0.31 

(6.3) 

32 

16 

2x  150 

8.9 

70 

1.030 

1.80 

0.13 

(7.3) 

13 

17 

2x150 

8.8 

160 

1.030 

3.60 

0.25 
(6.9) 

21 

18 

2  x  150 

8.6 

100 

1.018 

0.48 

0.03 
(6.2) 

5 

19 

2x150 

8.5 

100 

1.020 

3.25 

0.12 

(3.6) 

25 

*  Figures  in  brackets  indicate  percentages  of  total  nitrogen. 


which  accompanied  this  pathological  state.  The  observations 
noted  above  are  also  opposed  to  the  results  obtained  in  phosphorous 
poisoning15  a  condition  in  which  carbohydrate  is  almost  missing 
from  the  liver  and  blood.  On  the  other  hand,  hydrazine16  which 
behaves  in  a  manner  similar  to  phosphorus  'with  respect  to  its 
influence  upon  the  carbohydrate  of  the  organism  does  not  lead  to 
the  appearance  of  appreciable  quantities  of  lactic  acid  in  the  urine. 
From  these  contradictory  results  it  is  apparent  that  lactic  acid  must 
have  a  diverse  origin  under  the  different  conditions  mentioned. 
The  ammonia  content  of  the  urine  voided  by  the  dog  in  a  state  of 
inanition  was  not  greatly  influenced  by  the  cocaine  injections  and 
did  not  bear  a  direct  relationship  to  the  elimination  of  lactic  acid. 

16  Frank  and  Isaac:  Arch.  f.  exp.  Path.  u.  Pharm.,  lxiv,  p.  374,  1911. 
1GUnderhill:  This  Journal,  x,  p.  159,  1911. 


Frank  P.  Underbill  and  Clarence  L.  Black  251 


From  the  observations  here  recorded  the  conclusion  may  be 
drawn  that  the  appearance  of  lactic  acid  in  increased  quantity 
during  cocaine  poisoning  is  probably  associated  with  the  attendant 
increased  muscular  activity  induced  by  the  action  of  the  drug  upon 
the  nervous  system.  What  relation  augmented  lactic  acid  out- 
put bears  to  lack  of  oxygen  as  claimed  by  Araki  is  a  problem  dif- 
ficult of  decision  unless  one  accepts  the  view  put  forth  by  Feldman 
and  Hill17  that  increased  muscular  work  results  in  a  decreased 
amount  of  oxygen  in  the  muscles,  which  in  turn  causes  an  increased 
production  and  subsequent  excretion  of  lactic  acid. 

It  is  also  apparent  that  in  cocaine  poisoning  greater  quantities 
of  lactic  acid  are  eliminated  by  given  doses  of  cocaine  to  well-fed 
animals  than  occurs  under  the  same  conditions  during  an  interval 
of  starvation.  The  average  elimination  of  lactic  acid  during  co- 
caine poisoning  in  a  state  of  inanition  was  less  than  that  of  other 
animals  maintained  in  a  well-fed  condition,  but  without  cocaine 
administration.  It  seems  probable,  therefore,  that  during  cocaine 
poisoning,  carbohydrate  material  may  be  intimately  associated 
with  the  production  of  lactic  acid. 

conclusions. 

In  confirmation  of  previous  investigation,  it  is  found  that  co- 
caine introduced  subcutaneously  into  dogs  causes  a  temporary 
but  significant  increase  in  body  temperature. 

With  daily  doses  of  10  mgms.  of  cocaine  hydrochloride  per  kilo 
of  body  weight  for  short  periods  of  time  no  influence  can  be  de- 
tected upon  nitrogenous  metabolism  nor  upon  fat  utilization. 

Fat  utilization  is  slightly  impaired  and  body  weight  is  consider- 
ably decreased  when  daily  injections  of  15  mgms.  cocaine  are 
administered. 

When  the  dose  of  cocaine  is  increased  to  20  mgms.  per  kilo 
body  weight  per  day  a  distinct  lowering  of  both  nitrogen  and  fat 
utilization  is  noted.  This  may  be  accompanied  by  a  slight  nega- 
tive nitrogen  balance. 

,  Lactic  acid  excretion  in  the  urine  is  markedly  increased  in  well- 
fed  dogs  and  rabbits  as  a  result  of  cocaine  injection.    In  a  starving 

17  Feldman  and  Hill:  loc.  cit. 

TH  E  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  XI,  NO.  3. 


252        Influence  of  Cocaine  upon  Metabolism 


condition  the  dog  eliminates  less  lactic  acid  after  cocaine  injections 
than  is  excreted  by  the  normal  well-fed  animal. 

It  is  not  unlikely  that  the  increased  lactic  acid  elimination  after 
cocaine  injections  is  associated  with  increased  muscular  activity 
induced  by  the  drug. 

The  ammonia  output  apparently  bears  little  relation  to  lactic 
acid  elimination  under  the  experimental  conditions. 

Lactic  acid  and  carbohydrate  metabolism  are  presumably  inti- 
mately associated  although  there  are  indications  that  lactic  acid 
may  at  times  arise  from  more  than  a  single  antecedent. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  XI.  No.  5,  1912 


THE  PHYSIOLOGICAL  ACTION  OF  SOME  PYRIMIDINE 
COMPOUNDS  OF  THE  BARBITURIC  ACID  SERIES. 

By  ISRAEL  S.  KLEINER. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  March  27,  1912.) 

Aside  from  the  fact  that  certain  pyrimidine  compounds  are  con- 
stituents of  the  nucleic  acid  molecule,  their  possible  biochemical 
importance  is  attested  by  a  structural  relation  to  the  purines, 
creatine,  creatinine,  allantoi'n  and  other  compounds  of  physiologi- 
cal interest.  Moreover  a  few  pyrimidines  are  known  to  have  a 
marked  pharmacological  action. 

The  first  substance  of  this  type  used  in  physiological  experiments  was 
alloxantine,  which  is  formed  by  the  reduction  of  alloxan. 

HN— CO  HN— CO  OC—  NH 

I  I         +H2-        II  'I 

2       OC    CO      ^    2      OC    CH— O  C— OH  CO 

II  >         I      I                 I  I 
HN— CO  HN— CO  OC  NH 

Wohler  and  Frerichs1  fed  5  to  6  grams  of  this  to  men  but  could  not  recover 
any  in  the  urine;  nor  was  alloxan  found.  The  urine  was  rich  in  urea,  and 
a  breaking  down  of  alloxantine  to  urea  and  other  products  was  believed  to 
be  probable.  No  mention  is  made  of  any  toxic  effects,  although  if  any  had 
been  experienced  they  would  undoubtedly  have  been  described  because  the 
subjects  were  human  beings. 

Koehne2  fed  alloxan  and  alloxantine  in  8-gram  doses  to  dogs.  Each 
caused  a  mild  diarrhea  without  other  symptoms.  No  alloxan  or  alloxantine 
was  excreted  in  the  urine;  but  small  amounts  of  oxalic  and  parabanic  acids 
were  found.  Working  independently  of  Koehne  with  the  same  compounds 
Lusini3  obtained  results  different  in  some  respects  at  least.    In  his  experi- 


1  Wohler  and  Frerichs:  Ann.  d.  Chem.  u.  Pharm.,  lxv,  pp.  335-349,  1848. 

2  Koehne:  Inaugural  Dissertation,  Rostock,  1894,  39  pp. 

3  Lusini:  Ann.  di  chim.  e  di  farmacol.,  xxi,  pp.  145-160,  1895;  pp.  .  41-257; 
and  xxii,  pp.  341-351,  1895;  pp.  385-394:  from  Chem.  Centralbl.,  1895,  i,  p. 
1074;  ii,  p.  838. 

443 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  XI,  NO.  5 


444 


Action  of  Certain  Pyrimidines 


ments  he  found  that  both  of  these  substances  attacked  the  skin  of  frogs, 
dogs  and  rabbits.  Both  acted  upon  the  cerebro-spinal  centers,  this  action 
being  divided  into  two  periods,  (a)  hyper-reflex-excitability  followed  by 
rigidity,  and  (b)  hypo-reflex-excitability  and  paralysis.  This  second  stage 
had  previously  been  noted  by  Curci,4  who  because  of  the  use  of  a  larger 
dosage  had  overlooked  the  first  stage.  These  and  other  minor  effects  varied 
slightly  with  the  compound  used,  alloxan  being  in  general  more  toxic  than 
alloxantine.  Alloxantine  strongly  reduced  the  hemoglobin  of  the  blood 
both  in  vitro5  and  in  vivo.  Among  other  phenomena  produced  in  frogs  by 
alloxan,  mydriasis  is  noteworthy.  Alloxan  also  had  a  powerful  influence 
on  the  heart;  the  contractions  were  diminished  in  vigor,  diastolic  pauses 
lengthened,  and  finally  the  heart  stopped  in  diastole. 

According  to  Lusini,  alloxan  was  non-toxic  when  given  per  os,  0.5  gram 
being  easily  borne.  It  did  not  reappear  in  the  urine  but  parabanic  acid 
and  alloxantine  were  found.  When  Lusini  fed  alloxantine  he  recovered 
only  slight  traces  in  the  urine.  A  small  amount  of  dialuric  acid  was  found 
together  with  parabanic  acid  and  murexide  in  larger  quantities.  Lusini 

HN— 
I 

reached  the  conclusion  that  the  group,  OC     is  able  to  stimulate  and  then 

I 

.    '  HN — 

HN — CO 

inhibit  the  nerve  centers,  and  that  the  grouping,  q^L,  has  no  such  power. 

I 

It  is,  according  to  Lusini,  the  ketone-like  CO  which  seems  to  have  the  stimu- 
lating property,  and  the  abundance  of  these  groups  increases  the  toxicity 
of  alloxan. 

More  recently  Steudel6  has  attempted  to  ascertain  whether  pyrimidines 
may  be  built  up  to  purines  in  the  animal  body.  The  compounds  used  in- 
cluded those  which  Behrend  and  Roosen7  had  described  as  intermediate 
products  in  the  synthesis  of  uric  acid  in  the  chemical  laboratory.  At  the 
outset  it  may  be  stated  that  a  purine  synthesis  in  vivo  was  not  established. 
Steudel  fed  the  substances  to  a  bitch  weighing  6.2  kg.  in  doses  of  1  gram  per 
day  with  meat  and  attempted  to  isolate  them  or  their  purine  deriva- 
tives in  the  urine.  4-Methyluracil  and  5-nitrouracil  were  found  unchanged 
in  the  urine.  5-Nitrouracil-4-carboxylic  acid,  however,  did  not  reappear  in 
the  urine.  Steudel  believes  that  it  underwent  a  complete  decomposition 
in  the  organism,  although  he  does  not  consider  the  possibility  of  the  non- 
absorpti  n  from  the  alimentary  tract  and  does  not  report  any  analyses  of 


4  Curci:  Cited  by  Lusini:  Ann.  di  chim.  e  di  farmacol.,  xxi,  pp.  145-160, 
1895;  from  Chem.  Centralbl.,  1895,  i,  p.  1074. 

6  This  property  was  described  by  Kowalewsky:  Centralbl.  f.  d.  med.  Wis- 
sensch.,  xxv,  pp.  1-3,  17-18,  1887. 

6  Steudel:  Zeitschr.  f.  physiol.  Chem.,  xxxii,  pp.  285-290,  1901. 

7  Behrend  and  Roosen:  Ann.  d.  Chem.,  ccli,  pp.  235-256,  1889. 


Israel  S.  Kleiner 


445 


the  feces.  Of  the  following  pyrimidines  none  was  recovered  in  the  urine 
after  feeding,  nor  was  any  difficultly  soluble  condensation  product  detected : 
isobarbituric  acid,  isodialuric  acid,  thymine  and  uracil.  This  author  points 
out  the  striking  difference  in  behavior  between  thymine  (5-methyl-2,6- 
dioxypyrimidine)  and  4-methyluracil.  Structurally  they  differ  only  in  the 
position  of  the  methyl  group  but  the  former  is  broken  down  in  the  body 
while  the  latter  is  not.  If,  however,  a  nitro  group  is  substituted  for  the 
methyl  in  thymine  the  physiological  character  of  the  pyrimidine  is  reversed; 
for  it  now  passes  unchanged  through  the  kidney. 

Although  a  purine  synthesis  could  not  be  demonstrated,  Steudel  deter- 
mined to  extend  his  experiments  with  other  pyrimidines,  namely,  2,4-di- 

HN— CO 

I  I 

amino-6-oxypyrimidine,  H2N — C    CH  and  2,4,5-triamino-6-oxypy- 

II  II 

N— C— NH2 

HN— CO 

I  1 

rimidine,  H2NC    C — NH2  which  Traube8  had  obtained  as  intermediate 

II  II 

N— C— NH2 

products  in  his  synthesis  of  guanine.  Both  were  administered  as  the  sul- 
phates in  1-gram  dose  in  the  manner  above  described.  Both  were  reported 
to  be  toxic,  which  was  surprising  inasmuch  as  none  of  the  other  compounds 
had  been  accompanied  by  any  untoward  symptoms.  Feeding  of  the  2, 
4-diaminopyrimidine  was  followed  by  vomiting  and  the  triamino  compound 
provoked  equally  serious  disturbances.  About  an  hour  after  the  substance 
was  taken,  there  occurred  attempts  at  vomiting  without  any  vomitus  being 
ejected.  The  animal  had  no  appetite  and  lay  on  one  side  almost  all  day. 
The  urine  contained  protein,  hyaline  cylindroids  and  the  unchanged  tri- 
amino compound.  The  last  was  recovered  as  the  sulphate  and  identified 
by  the  violet  color  produced  by  saturating  it  with  ammonia.  By  subcu- 
taneous injections  the  lethal  dose  for  rats  was  determined  as  0.2  gram  for 
2,4-diamino-6-oxypyrimidine  sulphate  and  0.1  gram  for  2,4,5-triamino-6- 
oxypyrimidine  sulphate.  Autopsy  of  the  rats  poisoned  with  the  diamino 
substance  revealed  nothing  characteristic;  but  the  kidneys  of  the  animals 
which  had  received  the  triamino  compound  contained  numerous  concre- 
tions and  resembled  microscopically  the  kidneys  of  dogs  poisoned  with 
adenine.9 

From  these  results,  Steudel  concluded  that  the  attachment  of  amino  groups 
to  the  pyrimidine  ring  transforms  harmless,  indifferent  substances  into 
poisonous  ones.  The  toxicity  of  adenine,  6-aminopurine,  he  regards  as  an 
analogous  phenomenon  in  the  purine  series.  He  believes  that  an  examina- 
tion of  other  amino  derivatives  of  the  pyrimidine  and  purine  compounds 
will  prove  the  universality  of  this  law.    No  analyses  of  the  two  amino- 


8  Traube:  Ber.  d.  deutsch.  chem.  Gesellsch.,  xxxiii,  pp.  1371-1383,  1900. 

9  Minkowski:  Arch  f.  exp.  Path.  u.  Pharm.,  xli,  pp.  375-420,  1898. 


446 


Action  of  Certain  Pyrimidines 


pyrimidines  fed  are  presented  by  Steudel,  nor  are  any  data  as  to  their  solu- 
bility given. 

In  a  later  contribution  Steudel10  reported  the  investigation  of  other  mem- 
bers of  this  series.  Pseudo-uric  acid  and  isouric  acid  did  not  result  in  a 
purine  synthesis  although  both  have  been  transformed  into  uric  acid  in  vitro. 
A  similar  result  was  obtained  when  hydrouracil  was  fed.  2-Thio-4-methyl- 
uracil,  like  4-methyluracil  described  above,  was  quickly  excreted  in  the 
urine.  2-Amino-4-methyluracil,  which  differs  from  thiomethyluracil  only 
in  the  substitution  of  an  amino  group  for  sulphur  in  the  2-position  did  not 
appear  in  the  urine,  nor  was  any  other  characteristic  product  found.  Steu- 
del concludes  that  none  of  the  pyrimidines  used  by  him  are  adapted  to  a 
synthesis  of  purine  compounds  in  the  dog. 

The  pharmacological  action  of  some  pyrimidines  was  studied  by  Fischer 
and  von  Mering11  with  interesting  results.  They  discovered  that  certain 
alkyl  derivatives  possess  an  action  similar  to  that  of  sulphonal.  The  latter, 
diethylsulphone-dimethylmethane,  is  rich  in  alkyl  groups;  and  it  was  the 
idea  of  these  authors  to  experiment  with  other  alkyl  organic  compounds, 
many  of  which  Fischer  had  synthesized,  in  the  hope  of  ascertaining  the 
essential  or  most  effective  groupings  for  hypnotic  action.  Of  especial  inter- 
est are  the  cyclic  compounds  employed,  which  are  derivatives  of  barbituric 
acid  and  of  malonyl  guanidine.  It  was  found  that  the  5-monoalkyl  deriva- 
tives of  barbituric  acid  have  no  hypnotic  action,  nor  has  the  5,  5-dimethyl 
derivative;  but  when  both  hydrogen  atoms  in  the  5  position  are  replaced 
by  alkyl  groups,  at  least  one  being  higher  than  methyl,  the  compound 
acquires  sleep-producing  powers.  This  reaches  its  maximum  in  5,  5-dipro- 
pylbarbituric  acid.  Some  of  the  compounds  studied  proved  toxic;  for 
example,  substitution  of  the  H  in  the  1  position  by  CH3  or  of  the  O  in  the 
2  position  by  S  transformed  5,5-diethylbarbituric  acid  into  a  toxic  com- 
pound.    However,   5,5-dipropylmalonyl   guanidine,      HN — CO  as 


well  as  diethylmalonuric  acid,  H2N    COOH     had  no  pharmacological 


I      I  /C3H7 


HN  =  C  C< 

I      I  XC3H7 
HN— CO 


5,  5-Diethylbarbituric  acid,  HN — CO 

I      I  /C2H5 

oc  c< 

I      I  XC2H5 
HN— CO 


has  been  used  widely  in 


10  Steudel:  Zeitschr.  f.  physiol  Chem.,  xxxix,  pp.  136-142,  1903. 

11  Fischer  and  von  Mering:  Therapie  der  Gegenwart,  N.  F.,  v,  pp.  97-101, 
1903. 


Israel  S.  Kleiner 


447 


medicine  as  an  hypnotic  under  the  name  "veronal,"  its  sodium  salt  as 
"medinal,"  and  the  dipropyl  compound  to  a  less  extent  as  "proponal." 
Fischer  and  von  Mering12  have  found  that  most  of  the,  veronal  is  excreted 
from  the  body  unchanged.  Recently  P.  Fischer  and  Hoppe,13  Bachem,14 
Grober,15  and  Jacobj16  have  added  many  new  facts  to  the  literature  of 
veronal. 

Wolf17  injected  uracil,  thymine,  and  cytosine  in  10  to  50  mgs.  doses  into 
the  circulation  of  cats,  but  observed  no  effect  upon  arterial  pressure,  intesti- 
nal volume,  respiration,  or  rate  of  blood-clotting.  Sweet  and  Levene18  fed 
thymine  to  a  dog  with  an  Eck's  fistula  (on  the  basis  of  Steudel's  contention 
that  thymine  is  destroyed  by  the  normal  dog).  A  marked  diuresis  resulted 
and  thymine  was  found  in  the  urine  in  considerable  amount.  This  is  inter- 
esting in  view  of  the  close  relationship  between  this  methylated  pyrimidine 
and  the  methyl  substituted  xanthines:  theophylline,  theobromine,  and 
caffeine  which  are  also  diuretics. 

Mendel  and  Myers19  have  however  recently  shown  that  thymine  is  not 
completely  destroyed  normally  by  the  dog,  nor  is  uracil  nor  cytosine.  Diure- 
sis was  not  observed  by  them  after  the  administration  of  thymine.  The 
output  of  purines,  creatinine  and  urea  +  ammonia  was  not  influenced  by 
administering  any  of  these  to  rabbits,  dogs  or  men.  None  of  the  compounds 
had  any  marked  pharmacological  effects.  This  is  especially  interesting 
because  cytosine  is  an  amino-pyrimidine,  closely  related  to  the  compound 
alleged  by  Steudel  to  be  toxic. 

EXPERIMENTAL  PART.20 

Dogs,  rabbits,  and  guinea  pigs  were  used  in  the  physiological 
studies.  The  dogs  were  not  catheterized,  as  the  time  relations 
were  not  of  interest;  but  in  the  case  of  rabbits  the  urine  was 
removed  by  artificial  means  in  some  experiments.  The  dogs'  food 
always  was  mixed  with  bone  so  that  the  feces  became  firm  and  did 
not  contaminate  the  urine.  For  the  same  reason  the  rabbits  and 
guinea  pigs  were  given  some  grain  in  addition  to  carrots.  The 
compounds  were  administered  subcutaneously,  intraperitoneally, 
or  by  mouth. 

12  Fischer  and  von  Mering:  Therapie  der  Gegenwart,  April,  1904. 

13  P.  Fischer  and  Hoppe:  Miinahener  med.  Wochenschr.,  1909,  p.  1429. 

14  Bachem:  Arch.  f.  exp.  Path.  u.  Pharm.,  lxiii,  p.  228,  1910. 

15  Grober:  Bio'chem.  Zeitschr.,  xxxi,  p.  1,  1911. 

16  Jacobj:  Arch.  f.  exp.  Path.  u.  Pharm.,  lxvi,  p.  241,  1911. 

17  Wolf:  Journ.  of  Physiol.,  xxxii,  pp.  171-174,  1905. 

18  Sweet  and  Levene:  Journ.  of  Exp.  Med.,  ix,  pp.  229-239.  1907. 

19  Mendel  and  Myers:  Amer.  Journ.  of  Physiol.,  xxvi,  pp.  77-105,  1910. 

20  The  experimental  data  in  this  paper  are  taken  from  the  writer's  dis- 
sertation for  the  degree  of  Doctor  of  Philosophy,  Yale  University,  1909. 


448 


Action  of  Certain  Pyrimidines 


The  substances  used  were  barbituric  acid  and  its  amino-deriv- 


atives. 

HN— CO 

I  I 
OC  CH2 

I  I 
HN— CO 

Barbituric  acid 
(Malonyl  urea) 


HN— CO 

I  I 
HN  =  C  CH2 

I  I 
HN— CO 

Malonyl  guanidine 


HN— CO 


HN— CO 

HN  =  C  CHNH2 

I  I 
HN— CO 

5-Aminomalonyl 
guanidine 

HN— CO 


H2NC  CH 

II  II 

N— CNH2 

2,  4-Diamino-6-oxy- 
pyrimidine 


H2NC  CNH2 

N — CNH2 
2,  4,  5-Triamino-6-oxy- 
pyrimidine 


Barbituric  Acid. 

Barbituric  acid  was  made  essentially  according  to  Michael's21 
method,  the  principle  of  which  consists  in  condensing  urea  with 
diethylmalonate  in  the  presence  of  sodium  ethylate.  The  yield 
represented  71  per  cent  of  the  theoretical  and  the  acid  obtained 
gave  the  following  results  on  analysis  (Kjeldahl-Gunning method). 


N 


Calculated  for 
C4H4N2O3: 

21.87 


Found: 
21.87 

21. 6522 
21. 5322 


Barbituric  acid  crystallizes  in  two  forms,  the  anhydrous  as 
needles,  and  the  hydrated  as  rhombic  prisms.  It  is  slightly  soluble 
in  water.  A  rough  solubility  determination  showed  that  a  2.68 
per  cent  solution  can  be  prepared  at  40  to  43°. 

Efforts  to  obtain  a  method  for  estimating  barbituric  acid  quanti- 
tatively in  urine  were  unsuccessful,  although  a  qualitative  color 
test  afforded  a  means  of  getting  rough  values  colorimetrically. 
The  difficulty  lies  in  the  fact  that  many  of  the  properties  of  bar- 
bituric acid  are  possessed  also  by  hippuric  acid.    As  the  latter 

21  Michael:  Journ.  f.  prakt.  Chem.,  (2),  xxxv,  pp.  449-459,  1887. 

22  These  two  analyses  were  made  several  months  later. 


Israel  S.  Kleiner  449 


occurs  constantly  in  all  ordinary  urines,  and  in  considerable  amount 
in  the  urine  of  herbivora,  it  proved  an  effective  bar.  Both  com- 
pounds are  precipitated  by  mercuric  sulphate  and  by  silver  nitrate; 
both  are  soluble  in  ethyl  acetate  and  amyl  alcohol,  and  insoluble 
in  ligroin,  petroleum  ether  and  benzene.  The  color  reaction  referred 
to  is  based  on  Baeyer's23  observation  that  nitroso-barbituric  acid, 
in  the  presence  of  ferrous  acetate,  yields  a  deep  prussian  blue  color. 
The  directions  for  this  test  are  as  follows:  3  cc.  of  urine  are  treated 
with  three  drops  of  2  per  cent  sodium  nitrite  solution;  about  0.5  cc. 
of  10  per  cent  sulphuric  acid  is  added  and  the  solution  is  now  made 
alkaline  with  sodium  carbonate  solution;  on  addition  of  one  or  two 
drops  of  strong  ferrous  sulphate  solution  a  beautiful  blue  appears  in 
the  presence  of  barbituric  acid.  When  the  expression '  'NaN02-FeS04 
reaction"  is  employed  hereafter  it  will  be  understood  that  this  test 
is  meant.  Other  members  of  this  series  give  this  reaction  but  thy- 
mine, cytosine  and  uracil  do  not.24  Since  urine  frequently  assumes 
a  deeper  color  when  subjected  to  this  treatment,  a  direct  colori- 
metric  estimation  was  not  attempted  but  a  crude  method  was 
worked  out,  in  which  the  greatest  dilution  allowing  a  positive  test 
was  considered  the  standard.  It  was  thus  found  that  a  0.0023 
per  cent  solution  of  barbituric  acid  in  water  is  the  limit  for  this 
test,  and  hence  the  standard  for  comparison. 

Barbituric  acid  is  precipitated  by  mercuric  sulphate  solution. 
It  gives  Jaffe's  reaction  as  applied  to  creatinine.  A  red  color 
results  when  ferric  chloride  solution  is  added  to  barbituric  acid. 

The  sodium  salt  was  made  by  dissolving  the  acid  in  the  amount 
of  NaOH  calculated  to  form  the  disodium  salt,  concentrating  and 
allowing  the  salt  to  crystallize.  Needle  crystals  were  obtained; 
but  that  they  were  probably  a  mixture  of  the  mono-  and  disodium 
salts  is  evident  from  the  nitrogen  determination. 

23  Baeyer:  Ann.  d.  Chem.  u.  Pharm.,  cxxvii,  pp.  199-236,  1863. 

24  None  of  the  compounds  of  the  barbituric  acid  series  give  the  character- 
istic reactions  of  uracil,  thymine  or  cytosine.  For  example,  if  thymine  in 
substance  be  treated  with  diazobenzol-sulphonic  acid  a  reddish  purple  color 
results ;  tested  in  the  same  way  barbituric  acid  gives  a  red,  malonyl  guani- 
dine  and  cyanacetylguanidine  a  deep  orange  and  the  others  a  yellow  or  green 
color.  When  uracil  or  cytosine  is  dissolved  in  about  5  cc.  of  water,  bromine 
water  added  in  slight  excess  and  the  solution  boiled,  a  deep  purple  precip- 
itate results  on  the  addition  of  baryta  water.  None  of  the  barbituric  acid 
series  studied  gives  this  test. 


450 


Action  of  Certain  Pyrimidines 


N 


Calculated  for  Calculated  for 
C^NzOsNa*:  C^s^OsNa: 

. ..  16.28  18.66 


Found: 
17.58 


As  illustrations  of  the  general  method  employed  in  the  animal 
experiments  two  typical  protocols  will  first  be  given. 

Experiment  1.  A  rabbit  weighing  2  kg.  was  given  0.519  gram  barbituric 
acid  in  about  25  cc.  of  water  at  40°,  hypodermically.  The  urines  of  the  next 
two  days  were  precipitated  with  mercuric  sulphate,  the  precipitate  decom- 
posed with  hydrogen  sulphide  and,  after  removing  the  mercuric  sulphide, 
the  colorimetric  determination  made.  The  amount  excreted  was  estimated 
at  0.026  gram. 

No  hypnotic  or  toxic  action  was  exerted  by  the  compound.  Its  acidic 
character,  however,  made  it  harmful  to  the  tissues  at  the  point  of  injection; 
this  caused  an  opening  in  the  body  wall  which  led  to  the  death  of  the  animal 
seven  days  after  the  injection. 

Experiment  17.  0.64  gram  of  sodium  barbiturate  in  45  cc.  water  contain- 
ing 0.1  cc.  &  NaOH  at  38°  were  injected  intraperitoneal!}^  into  a  rabbit 
weighing  1.6  kg.    Diarrhea  resulted  in  about  two  hours  and  this  condition 
persisted  for  five  days.    The  urines  of  the  first  two  days  gave  positive 
NaNCVFeSCX  tests  and  these  corresponded  to  0.04  gram  of  barbituric  acid. 

These  as  well  as  other  experiments  with  barbituric  acid  are  tabulated 
on  the  opposite  page. 

From  this  table  it  is  seen  that  the  fatal  termination  of 'Experi- 
ments 1  and  3  must  be  ascribed  to  the  acidic  properties  of  barbi- 
turic acid;  for  when  larger  amounts  of  the  sodium  salt  were  given 
as  in  Experiments  12  and  17  no  toxic  effects  resulted.  The  only 
physiological  effect,  which  may  be  ascribed  to  its  structure,  is  its 
diarrheal  action ;  but  a  greater  number  of  experiments  need  to  be 
done  to  settle  this  point.  In  this  connection  it  is  interesting  to 
recall  the  fact,  noted  above,  that  Koehne25  observed  a  mild  diar- 
rhea after  feeding  alloxan  and  alloxantine.  Again,  the  fact  that 
barbituric  acid  has  no  hypnotic  action  harmonizes  with  Fischer 
and  von  Mering's26  experiments  on  substituted  barbituric  acids, 
in  which,  as  detailed  above,  they  found  that  the  lower  the  substi- 
tuted alkyl  groups,  the  less  hypnotic  the  influence  possessed  by 
the  complex.  In  barbituric  acid,  the  lowest  degree  is  reached  and 
no  hypnotic  action  is  observed. 


25  Koehne:  Inaugural  Dissertation,  Rostock,  1894. 

26  Fischer  and  von  Mering:  Therapie  der  Gegenwart,  v,  pp.  97-101,  1903. 


Israel  S.  Kleiner 


451 


Animal  Experiments. 


TABLE  I. 

Barbituric  Acid  (Malonyl  urea). 


NUMBER  AND 
ANIMAL 


(1)  Rabbit 


(2)  Rabbit. 

(3)  Rabbit. 


(4)  Rabbit. 


(6)  Rabbit, 


(12)  Guinea 
pig.. 

(17)  Rabbit. 


AMOUNT  GIVEN 


Total 


kg.     \  gram 

2.0  !  0.52 


2.1  i  0.32 


2.2  [  0.53 


1.9 


0.2 


1.9  0.6 


0.5 


1.6 


0.3 


0.64 


Per 
kilo- 
gram 


MODE  OF 
ADMINISTRATION 


gram 

0.26  Subcutaneously 


0.15  Subcutaneously 


0.24  Intraperitoneally 


0.10  Intraperitoneally 


0.3     Per  os.. 


0.6  Subcutaneously 


0.4  Intraperitoneally 


REMARKS  AND  RESULTS 


Not  toxic  except  for 
necrosis  at  point 
of  injection,  which 
caused  death  seven 
days  later.  Some 
excreted. 

Not  toxic.  Excret- 
ed about  one-third 
(?) 

Death  in  three  days. 

.  Diarrhea  at  first. 
Diminished  flow  of 
urine  (28  cc.  in  two 
days)  containing 
0.09  gram  (?). Au- 
topsy revealed  fib- 
rinous adhesions  in 
peritoneal  cavity. 

Recrystallized  prep- 
aration used.  Not 
toxic.  Under  ob- 
servation fifty-six 
days. 

Marked  diarrhea. 
Excreted  about  -jV 
in  urine. 

Na  salt  used.  Not 
toxic.  Excreted 
0.01  gram.  (?) 

Na  salt  used.  Ex- 
creted about  0.04 
gram  (?)  Diar- 
rhea for  five  days, 
otherwise  not  tox- 
ic. Under  obser- 
vation thirty-one 
days. 


452 


Action  of  Certain  Pyrimidines 


Malonyl  Guanidine. 

In  synthesizing  malonyl  guanidine  Michael's27  procedure  was 
essentially  followed.  The  pyrimidine  was  obtained  in  the  form  of 
its  sodium  salt  which  was  dissolved  in  water  and  dilute  NaOH,and 
the  free  pyrimidine  precipitated  with  acetic  acid.  Malonyl  guani- 
dine crystallized  in  fine  white  needles  which,  after  drying  in  a 
desiccator,  were  analyzed  for  nitrogen. 

Calculated  for 
C4H6N,02+H20:   C4H6N3O2:  Found: 

N   28.96         33.07  32.05 

31.91 

The  low  nitrogen  values  are  probably  due  to  incomplete  removal 
of  the  water  of  crystallization  by  simple  desiccation.  Inasmuch 
as  the  analysis  was  fairly  close  and  the  preparation  was  pure  white 
no  further  purification  was  attempted.  It  was  only  slightly  solu- 
ble in  water.  At  40  to  43°  a  0.049  per  cent  solution  was  the  strong- 
est obtainable.  This,  of  course,  renders  malonyl  guanidine  itself 
unsuitable  for  injection  experiments  and  the  sodium  salt  was 
accordingly  used.  In  preparing  this,  the  pyrimidine  was  dissolved 
in  NaOH,  as  little  in  excess  of  the  calculated  amount  as  would 
bring  about  solution  being  used.  On  concentration,  fine  pale  pink 
needles  crystallized  out.  From  the  analyses,  which  follow,  this 
salt  must  contain  four  molecules  of  water  of  crystallization  which 
are  lost  in  the  desiccator  very  slowly. 

Calculated  for  Found : 

C4H4NaN302+4H20:  (air-dry) 

N   19.00  18.77 

Calculated  for  Found: 
C4H4NaN802:  (desiccated) 
N   28.19  25.31 

A  method  for  recovering  malonyl  guanidine  from  urine  is  at 
once  suggested  by  the  slight  solubility  of 'the  free  substance. 
However,  if  urine  is  acidified  and  allowed  to  stand,  uric  acid 
and,  if  concentrated  sufficiently,  hippuric  acid  will  also  crystallize 
out.  The  NaN02-FeS04  reaction  described  above  for  barbituric 
acid  is  also  applicable  to  malonyl  guanidine.  The  limit  for  this 
test  in  urine  is  0.004  per  cent.  Another  mode  of  estimation  by 
means  of  this  color  reaction  was  tried  as  follows:  0.002  gram  in 

27  Michael:  Journ.  f.  prakt.  Chem.,  xlix,  pp.  26-43,  1894. 


Israel  S.  Kleiner 


453 


3  cc.  water  was  converted  to  the  prussian  blue  compound  and  dilu- 
tions made  until  the  blue  was  no  longer  distinctly  discernible  in 
a  100  cc.  cylinder.  The  concentration  just  above  this  was  con- 
sidered the  standard.  By  such  a  rough  method  it  was  found  that 
a  distinct  blue  can  be  seen  when  there  is  an  amount  corresponding 
to  0.0004  per  cent  present. 

Sodium  malonyl  guanidine  is  not  precipitated  by  ammoniacal 
silver  nitrate  solution,  but  is  precipitated  quantitatively  by  mer- 
curic sulphate  solution.  With  picric  acid  and  alkali  a  red  color  is 
formed  as  in  Jaffe's  test  for  creatinine. 

From  the  animal  experiments  (see  Table  II)  it  is  seen  that  mal- 
onyl guanidine  is  non-toxic,  at  least  in  the  doses  for  the  animals 
used.  The  failure  to  detect  the  substance  or  a  related  compound 
in  the  urine  of  Experiment  5  may  be  due  to  the  small  amount 
injected. 

TABLE  II. 

Animal  Experiments.    Malonyl  Guanidine. 


NUMBER  AND 
ANIMAL 


(5)  Rabbit .  .  . 
(9)  Rabbit. .  . 
(26)  Rabbit .  . 


(24)  Dog. 


kg. 
2.2 


1.9 


2.1 


10. 


AMOUNT  GIVEN 


Total 


gram 

0.09 


0.41 


0.22 


2.1 


Per 
kilo- 
gram 


gram 

0.04 


0.21 


0.10 


0.21 


MODE  OF 
ADMINISTRATION 


REMARKS  AND  RESULTS 


Subcutaneously 


Subcutaneously 


Subcutaneously 


Per  os 


Sodium  salt  used. 
No  effects.  Not 
detected  in  urine. 

Sodium  salt  used. 
No  effects.  De- 
tected in  urine. 

Sodium  salt  used. 
Mild  diarrhea;  no 
other  effects.  All 
(?)  excreted  in 
urine. 

Free  malonyl  guani- 
dine used.  Some 
absorbed  and  ex- 
creted, in  urine. 
No  toxic  effects. 


454  Action  of  Certain  Pyrimidines 


5-Aminomalonyl  Guanidine. 


This  compound  is  quite  difficult  to  obtain  in  good  yield  as  it 
decomposes  very  easily.  The  most  advantageous  method  was 
found  to  be  a  modification  of  one  described  by  Traube28  in  which 
the  sulphate  of  this  compound  can  be  prepared  directly  from  mal- 
onyl  guanidine.  The  directions  of  Traube  were  followed  as  far 
as  the  formation  of  5-aminomalonyl  guanidine  sulphate  by  reduc- 
tion with  H2S,  but  instead  of  extracting  this  salt  with  hot  water, 
the  sulphur  was  removed  by  means  of  CS2  and  the  sulphate  con- 
verted into  the  hydrochloride  by  treatment  with  BaCl2.  Traube's 
suggestion  of  adding  alcohol  to  induce  crystallization  was  not  found 
to  be  advantageous  since  the  crystals,  when  finally  obtained,  had 
a  pink  tinge.  Consequently,  the  fluid  was  concentrated  under 
diminished  pressure  and  allowed  to  crystallize.  Light  yellow  ros- 
ettes of  needles  formed  very  slowly. 

Analysis  of  this  preparation  (A)  by  the  Kjeldahl-Gunning  method 
showed  that,  in  spite  of  the  tinge  of  yellow  color,  the  salt  was  quite 
pure.  Another  preparation  (B)  made  by  the  same  method  gave 
a  higher  nitrogen  percentage. 


Its  solution,  which  is  acid  to  litmus,  very  quickly  turns  red,  owing 
undoubtedly  to  a  slight  oxidation.  It  stains  the  tissues  red  and 
has  a  faint  disagreeable  odor.  Boiling  with  NH4OH  yields  a  solu- 
tion colored  like  potassium  permanganate  and  this  changes  to  dark 
blue  on  addition  of  KOH.  It  will  give  the  NaN02-FeS04  reaction, 
but  not  readily  or  brilliantly.  The  Jaffe  color  reaction  for  creatin- 
ine is  not  given  by  this  salt.  It  is  precipitated  both  by  ammoni- 
acal  silver  nitrate  and  mercuric  sulphate,  but  as  very  little  can  be 
injected  into  an  animal  and  as  it  was  found  to  be  toxic  no  attempt 
was  made  to  isolate  it  from  urine. 

The  toxicity  of  this  compound  is  shown  in  the  following  illus- 
trative protocols  and  the  accompanying  table  (Table  III)  which 
summarizes  all  the  experiments.  The  hydrochloride  was  used  in 
each  case. 


Calculated  for 
(C4H6N402)HC1+H20: 


Found: 

28.55 
28.08 
29.41 


N 


28.57 


28  Traube:  Ber.  d.  deutsch.  chem.  Gesellsch.,  xxvi,  pp.  2551-2558,  1893. 


Israel  S.  Kleiner 


455 


Experiment  7.  November  30.  3:10  p.m.  A  rabbit  weighing  1.4  kg.  was 
given  subcutaneously  0.37  gram  in  35  cc.  water. 

3:20  p.m.  Has  defecated  very  soft  stools.    Moves  around  restlessly. 
4:15  p.m.,  4:50  p.m.  Apparently  well. 

December  1.  8:45  a.m.  Rabbit  found  dead.  Autopsy:  kidneys  are  very 
light  colored;  intestines  intensely  reddened;  liver,  light  brown;  large 
amount  of  bloody  fluid  in  peritoneal  cavity. 

Experiment  23.  March  11.  11 :00  a.m.  A  guinea  pig  weighing  450  grams 
was  given  0.036  gram  of  the  salt  in  about  10  cc.  water  subcutaneously. 

March  12.  2:15  p.m.  Has  eaten  60  grams  carrots  and  9  grams  oats. 
Urine,  64  cc,  alkaline; specific  gravity,  1.017;  albumin  present,  but  no  casts. 

March  13.  2:40  p.m.  Has  eaten  70  grams  carrots  and  3  grams  oats. 
Urine,  43  cc;  alkaline;  specific  gravity,  1.024;  large  amount  of  albumin; 
granular,  granular  partly  hyaline,  and  cellular  casts  found;  NaNCVFeSC^ 
test  negative. 

March  14.  2 :35  p.m.  Has  eaten  85  grams  carrots  and  4  grams  oats.  Urine 
30  cc;  alkaline;  albumin  present;  casts. 

March  15.  2:50  p.m.  Has  eaten  93  grams  carrots  and  3  grams  oats.  Urine 
57  cc;  alkaline;  specific  gravity,  1.021;  albumin;  casts. 

March  16.  8:40  a.m.  Animal  appears  well.  Weight  390  grams.  The 
animal  daily  ate  more  food  until  March  20,  when  the  usual  amount  (150 
grams  carrots  and  15  grams  oats)  was  entirely  consumed.  On  March  18, 
its  weight  had  dropped  to  360  gram  but  then  rose  to  440  grams  on  March  24. 
The  urine  still  contained  a  trace  of  albumin.  On  April  3 — twenty-three 
days  after  the  injection — the  animal  was  still  living  and  apparently  well. 

Experiment  22.  March  11.  4:00  p.m.  A  female  rabbit  weighing  2.44  kg. 
was  given  a  subcutaneous  injection  of  0.19  gram  in  about  40  cc.  water. 

March  12.  9:00  a.m.  Stools  partly  diarrheal.  2:15  p.m.  No  urine.  Has 
eaten  145  grams  carrots  but  no  oats. 

March  13.  2:40  p.m.  No  urine.  Has  eaten  130  grams  carrots  but  no 
oats. 

March  14.  11:00  a.m.  No  urine,  no  feces.  Has  eaten  85  grams  carrots 
and  8  grams  oats. 

March  15.  2:50  p.m.  Has  eaten  46  grams  carrots  but  no  oats.  Urine, 
163  cc;  alkaline;  specific  gravity,  1.013;  NaN02-FeS04  test  negative;  albu- 
min and  granular  casts  present;  slight  reduction  of  alkaline  copper  solu- 
tion (after  removing  albumin). 

March  16.  8:40  a.m.  Has  eaten  35  grams  carrots.  Animal  is  very  weak, 
breathes  slowly  and  can  not  hold  its  head  up. 

10:05  a.m.  Breathes  more  quickly  but  head  is  on  floor  of  the  cage. 

11:50  a.m.  Still  breathing;  extremely  weak. 

2:10  p.m.  Found  dead.  Urine,  52  cc;  alkaline;  specific  gravity,  1.010; 
albumin  and  casts  present;  reduction  positive. 

Autopsy.  Weight  2.26  kg.  All  viscera  hyperemic;  blood  of  liver  does 
not  clot  readily;  kidneys  edematous;  bladder  empty;  animal  is  quite  fat. 
Sections  of  tissues  preserved. 


456  Action  of  Certain  Pyrimidines 

table  nr. 

Animal  Experiments.    5-Aminomalonyl  guanidine. 


NUMBER  AND 
ANIMAL 


(7)  Rabbit.. 
(22)  Rabbit. 

(8)  Rabbit.. 


(10)  Guinea 
pig.. 


(27)  Guinea 
pig-- 


(25)  Guinea 
pig... 


(23)  Guinea 
pig... 


AMOUNT  GIVEN 


kg. 

1.4 
2.4 


2.6 


0.41 


0.54 


0.54 


0.45 


Total 


gram 

0.37 


0.19 


0.11 


0.05 


0.061 


0.048 


Per 
kilo- 
gram 


gram 

0.26 


0.08 


0.04 


0.12 


0.11 


0.09 


0.036  0.08 


MODE  OF 
ADMINISTRATION 


Subcutaneously 
Subcutaneously 

Subcutaneously 


Subcutaneously 


Subcutaneously 


Subcutaneously 


Subcutaneously 


REMARKS  AND  RESULTS 


Fatal  in  less  than 
eighteen  hours. 

Albuminiuria;  casts; 
glycosuria.  Death 
in  five  days. 

Fifty-three  cubic 
centimeters  urine 
in  first  forty-eight 
hours.  Albumi- 
nuria until  fourth 
day.  No  glyco- 
suria. Recovery. 

Albuminuria.  Death 
in  four  days.  Au- 
topsy: organs  ap- 
pear normal.  Blood 
does  not  clot  read- 

iiy. 

Not  fed  on  day  of 
injection.  Albu- 
minuria: mucus 
cylindroid  seen. 
Fatal  in  less  than 
two  days.  Autop- 
sy: one  fetus  pres- 
ent; large  amount 
of  bloody  subcu- 
taneous effusion. 
Kidneys  seem  con- 
tracted. 

Albuminuria  for  at 
least  seven  days; 
casts  and  leucocy- 
tes in  urine;  recov- 
ery; under  obser- 
vation twelve  days . 

Albuminuria;  casts; 
recovery. 


Israel  S.  Kleiner  457 


TABLE  III— Continued. 


NUMBER  AND 
ANIMAL 

AMOUNT  GIVEN 

MODE  OF 
ADMINISTR  \TION 

WEIGHT 

Total 

Per 
kilo- 
gram 

REMARKS  AND  RESULTS 

(21)  Guinea 
pig... 

kg. 

1.7 
0.52 

gram 

0.18 
0.04 

gram 

0.10 
0.08 

Per  os 
Per  os 

NTn    svmntoms'  no 

albuminuria.  Un- 
der observation 
eighteen  days. 
No    symptoms ;  n  0 
albuminuria.  Un- 
der observation 
thirty  days. 

From  these  results  it  appears  that  a  lethal  subcutaneous  dose 
for  rabbits  is  0.08  gram  per  kg.  and  for  guinea  pigs  0.11  gram  per 
kg.  It  is  also  evident  that  when  the  compound  is  fed  it  is  not 
toxic.  In  Experiment  20,  the  urine  was  repeatedly  examined  for 
substances  giving  the  NaNCVFeSC^  test  but  with  negative  results. 
The  feces,  however,  in  both  Experiments  20  and  21  were  tinged 
with  pink  at  times.  Probably  not  enough  of  the  compound  is 
absorbed  from  the  alimentary  tract  at  one  time  to  prove  toxic;  it 
may  be  mentioned,  however,  that  the  hydrochloride  is  fairly  solu- 
ble. That  the  compound  acts  mainly  on  the  kidneys  is  evident 
from  the  protocols  and  the  table,  but  substantiating  evidence  is 
given  by  the  histological  examination,  made  by  Professor  H.  Gideon 
Wells  to  whom  I  am  greatly  indebted  for  the  following  report. 

Experiment  22 — Rabbit.  Kidney.  Shows  extensive  necrosis  of  the  con- 
voluted tubules,  perhaps  one-fourth  of  the  tubules  seen  in  section  showing 
total  necrosis  of  the  epithelium.  The  necrotic  epithelium  desquamates 
into  the  lumen  of  the  tubule  which  it  fills  up,  and  all  stages  of  transition 
from  masses  of  necrotic  epithelium  to  granular  and  hyaline  casts  which  pack 
the  collecting  tubules  can  readily  be  made  out.  These  casts,  being  very 
abundant  and  staining  intensely  with  eosin,  give  the  sections  a  striking 
appearance.  The  tubular  epithelium  where  not  necrotic  is  strikingly  little 
altered,  some  tendency  to  vacuolization  of  the  cytoplasm  being  the  chief 
abnormality  noted.  Glomerules  congested,  swollen,  and  in  some  a  little 
granular  material  and  occasional  red  corpuscles  free  in  the  space  outside 
the  tuft;  in  general  the  glomerules  show  relatively  little  change.  There 
is  an  occasional  small  area  of  interstitial  hemorrhage.  To  summarize,  the 
poison  has  caused  a  marked  necrosis  of  the  epithelium  of  the  convoluted 


458 


Action  of  Certain  Pyrimidines 


tubules,  but  without  affecting  other  renal  structures  to  any  considerable 
degree. 

Liver.  No  definite  changes  except  the  accumulation  of  masses  of  yellow- 
ish brown  pigment  in  many  of  the  stellate  cells. 

Spleen.  Some  of  the  endothelial  cells  of  the  splenic  sinuses  contain 
brownish  pigment,  otherwise  no  change.  The  pigmentation  of  the  liver  and 
spleen  suggests  a  hemolytic  action  by  the  poison. 

Experiment  27 — Guinea  Pig.  Kidney.  Shows  the  same  necrosis  of  the 
secretory  epithelium  of  the  tubules  and  the  same  formation  of  casts  as 
described  in  Rabbit  22,  but  very  much  less  marked,  only  occasional  tubules 
showing  the  lesion. 

Liver.    No  pigmentation  or  other  distinct  changes. 

Spleen.    Much  more  pigment  than  in  Rabbit  22.    No  other  changes. 

Adrenal.    No  changes. 

Experiment  10 — Guinea  Pig.  Kidney.  Granular  and  hyaline  casts  are 
very  abundant  and  conspicuous,  although  there  are  fewer  tubules  showing 
necrosis  than  in  either  of  the  other  specimens.  When  found  it  is  typical, 
exactly  the  same  in  appearance  as  in  22  and  27.  The  casts  much  more  often 
show  desquamated  epithelial  cells  within  them.  Marked  congestion,  but 
no  other  changes.  The  constancy  of  the  finding  of  necrotic  tubular  epithe- 
lium in  all  three  kidneys  is  conclusive  evidence  that  this  is  a  specific  effect 
of  the  poison  given. 

Liver.    No  distinct  alterations. 

2,4-Diamino-6-oxypyrimidine. 

Both  2,4-diamino-6-oxypyrimidine  and  its  precursor,  cyana- 
cetylguanidine, were  used  in  the  experiments  on  animals.  They 
were  made  by  Traube's29  method  with  some  modifications.  Guani- 
dine  hydrochloride,  according  to  this  procedure,  is  condensed  with 
cyanethylacetate  forming,  in  part,  the  pyrimidine;  but  mainly 
cyanacetylguanidine,  which  is  easily  converted  into  the  pyrimi- 
dine by  alkali. 

H2N  COOC2H5 

I  I 
HN  =  C  +  CH2 

I  I 
H2N  CN 

The  yield  of  cyanacetylguanidine  was  35.8  per  cent  of  the  theo- 
retical, if  this  were  the  sole  end-product.  The  mother-liquor  was 
of  a  dark  red  color  and  on  concentration  yielded  a  large  amount 


HN— CO  HN— CO 


->     HNC    CH2  >     H2NC  CH2 

II  II  I 

H2N    CN  N— CNH 


29  Traube:  Ber.  d.  deutsch.  chem.  Gesellsch.,  xxxiii,  pp.  1371-1383,  1900. 


Israel  S.  Kleiner 


459 


of  material  which  was  used  in  the  preparation  Of  the  pyrimidine. 
The  first  crop  was  recrystallized  from  hot  water,  pulverized  and 
desiccated.  To  determine  whether  the  substance  obtained  was 
cyanacetylguanidine  or  the  pyrimidine,  advantage  was  taken  of 
the  fact  that  the  latter  crystallizes  with  one  molecule  of  water  of 
crystallization  while  the  former  is  water-free. 

Calculated  for 
(C4H6N40)+H20:    C4H6N40:  Found: 

H20   12.5  0.0  1.2 

This  preparation  was  consequently  cyanacetylguanidine  with 
very  little,  if  any,  pyrimidine  admixture.  Nitrogen  determina- 
tions by  the  Kjeldahl-Gunning  method  gave  low  figures,  perhaps 
because  some  HCN  may  have  been  formed  and  lost  or  because  of 
a  very  slight  admixture  of  the  pyrimidine. 

Calculated  for 
C4H6N4O:  Found: 

N   44.44  41.99 

41.96 

A  much  better  yield  is  obtained  by  using  guanidine  sulphocy- 
anide  in  place  of  the  hydrochloride,  as  the  mother  liquor  in  this 
case  is  not  as  dark  colored  and  may  be  evaporated  to  dryness 
without  much  loss  of  material.  In  this  modification,  when  used 
as  a  step  in  the  preparation  of  the  pyrimidine,  it  is  not  necessary 
to  remove  the  NaSCN  formed  until  the  2,4-diamino-6-oxypy- 
rimidine  is  precipitated  as  the  sulphate,  since  the  latter  can  be 
washed  free  from  inorganic  salts  with  water. 

Cyanacetylguanidine  is  quite  soluble  in  water — a  2.5  per  cent 
solution  being  easily  maintained  at  40° — and  is  suitable  for  injec- 
tion. Cyanacetylguanidine  forms  a  rose  red  isonitroso  compound 
(or  is  converted  into  the  isonitroso  derivative  of  2,4-diamino-6- 
oxypyrimidine)  on  adding  NaN02  and  H2S04  to  its  solution;  as 
this  is  quite  insoluble  it  may  be  isolated  from  the  urine.  Accord- 
ing to  Traube  the  isonitroso  compound  has  an  intense  yellow  or 
yellowish  green  color;  however,  with  our  preparation  the  brilliant 
red  compound  formed  first  and  did  not  become  yellow  until  addi- 
tional acid  was  used.  The  color  test  with  NaN02  and  FeS04  as 
described  above  is  also  positive  for  cyanacetylguanidine. 

For  the  transformation  of  cyanacetylguanidine  into  its  isomer, 
2,4-diamino-6-oxypyrimidine,  it  was  put  into  boiling  2-5  per  cent 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  XI,  NO.  5. 


460 


Action  of  Certain  Pyrimidines 


NaOH,  animal  charcoal  added,  boiled  a  few  minutes,  and  filtered 
into  a  beaker  placed  in  an  ice-bath.  As  some  NH3  is  split  off  by- 
boiling  with  alkali  in  this  way,  the  operation  must  necessarily  be 
performed  quickly.  The  solution  was  now  made  weakly  acid  with 
H2SO4  and  white  or  yellowish  needle-like  crystals  of  the  sulphate  of 
the  pyrimidine  appeared.  When  recrystallized  from  hot  water 
large  silky,  grayish  needles  were  obtained  and  these  were  again 
recrystallized  from  water  in  the  presence  of  dilute  H2S04  and  some 
charcoal;  the  crystals  resulting  were  of  a  light  yellow,  almost 
white  color. 

According  to  Traube  the  sulphate,  when  recrystallized  from 
water,  contains  one  molecule  of  water  of  crystallization  which  is 
not  driven  off  at  100°.  Its  composition  is  (C4H6N402).  H2S04-|- 
H20.  Our  preparation  agreed  in  its  nitrogen  content  with  this 
formula,  as  the  following  analyses  indicate. 

Calculated  for 
(C4H6N40)2-H2S04+H20:  Found: 

N   30.43  30.42 

30.27 

This  salt  is  sparingly  soluble  in  water.  A  rough  solubility  de- 
termination showed  that  at  43°  a  greater  concentration  than  0.49 
per  cent  could  not  be  maintained  and  at  a  slightly  lower  tempera- 
ture much  of  the  substance  instantly  crystallized  out.  An  aqueous 
solution  gives  a  positive  NaN02-FeS04  test. 

Because  of  the  poor  solubility  no  injection  experiments  were 
performed.  However,  Steudel's30  experiment,  in  which  he  reports 
this  compound  toxic  when  fed  to  a  dog,  was  repeated  in  exactly 
the  same  manner  and  with  the  same  relative  dosage. 

Expeeiment  15.  February  15.  10:20  a.m.  Bitch  weighing  9.6  kg.  fed 
180  grams  chopped  meat  with  bone  meal,  to  which  was  added  1.55  grams  of 
the  sulphate  of  the  pyrimidine. 

10:20  to  11:40  a.m.  Under  observation  almost  continually.  The  animal, 
which  has  always  been  playful,  shows  no  unusual  behavior,  but  is  appar- 
ently normal. 

2 :00  p.m.  Animal  still  lively. 

2:15  p.m.  Ate  some  meat  and  drank  water.    No  nausea  observed. 
5 :10  to  5 :20  p.m.  Animal  well. 

February  16.  9:00  a.m.  Fed  meat,  cracker  and  bone  meal.  Urine,  170 
cc. ;  specific  gravity,  1.055;  acid;  no  albumin.    On  adding  NaNC>2  and  H2S04 


30Steudel:  Zeitschr.  f.  physiol.  Chem.,  xxxii,  pp.  285-290,  1901. 


Israel  S.  Kleiner 


46i 


a  rose-colored  precipitate  appeared  which  was  filtered  off  and  washed  with 
hot  water,  alcohol  and  ether.  For  the  total  volume  of  urine  this  would  have 
amounted  to  1.17  grams.  It  was  dissolved  in  KOH,  reprecipitated  by  HC1, 
filtered  etc.  and  analyzed  by  the  Kjeldahl-Gunning  method  (for  nitrates). 

Calculated  for  isonltroso 
derivative  of 
2,4-diamino-6-oxy-  monamino-dioxy- 
pyrimidlne  pyrimidine 
(  =  C4HBN502):         (  =  dH4N408):  Found: 

N   45.16  35.89  39.25 

39.56 

Feeding  suspensions  of  the  salt  to  a  guinea  pig  and  to  a  rabbit  gave  simi- 
lar non-toxic  results  (see  Table  IV).  The  sulphate,  as  in  Steudel's  investi- 
gation, was  used  in  every  case. 

TABLE  IV. 


Animal  Experiments:    2, 4-Diamino-6-oxy  pyrimidine. 


NUMBER  AND 
ANIMAL 

AMOUNT  GIVEN 

MANNER  OF 
ADMINISTRATION 

WEIGHT 

Total 

Per 
kilo- 
gram 

REMARKS  AND  RESULTS 

kg. 

gram 

gram 

(15)  Dog  

9.6 

1.55 

0.16 

Per  os 

No  toxic  effect. Large 

(18)  Guinea 

proportion  excret- 
ed; deaminized  (?) 

pig.... 

0.16 

0.14 

0.87 

Per  os 

Fed  in  saccharose 
suspension  from 
pipette.  No  symp- 
toms. Under  ob- 
servation seven 
days. 

(19)  Rabbit.. 

1.96 

0.51 

0.26 

Per  os 

Given  in  suspen- 
sion in  water.  No 
albuminuria.  Na- 
N02  -  Fe  S04  test 
positive.  Unable  to 
obtain  an  isonitro- 
so  compound  as  in 
Experiment  15. 

It  is  therefore  evident  that  his  pyrimidine  is  not  toxic  when  given 
per  os  as  the  sulphate.  Doses  larger  than  those  reported  toxic  by 
Steudel  were  without  effect  upon  the  rabbit  and  guinea  pig  as 
Experiments  18  and  19  indicate. 


462 


Action  of  Certain  Pyrimidines 


2 ,4,5 -Triamino-6-oxy  pyrimidine. 


This  pyrimidine  was  prepared  according  to  Traube's31  directions 
and  was  isolated  as  the  sulphate.  When  crystallized  quickly  the 
salt  appears  as  small  rods  or  rectangular  prisms  but  if  allowed  to 
crystallize  slowly  large  needles  are  formed.  After  desiccation,  an 
analysis  gave  the  following  results. 


The  solubility  of  this  salt  is  about  the  same  as  that  of  the  diamino 
compound,  i.e.,  it  was  found  to  be  possible  to  obtain  a  0.49  per  cent 
solution  at  43°.  In  this  case,  however,  the  fluid  became  dark  dur- 
ing the  manipulation  and,  after  drying,  the  residue  was  dark  brown 
in  color.  It  is  thus  evident  that  some  chemical  change — a  decom- 
position or  oxidation — occurred  and  hence  the  determination  can 
only  be  regarded  as  an  evidence  of  the  very  slight  solubility  of 
the  substance  at  low  temperatures  and  of  its  instability,  when  in 
solution,  at  a  high  temperature. 

According  to  Traube,  if  an  ammoniacal  solution  of  the  sulphate 
be  shaken  so  as  to  afford  contact  with  the  air  the  fluid  assumes  an 
intense  violet  color  resembling  permanganate  solution.  This  re- 
action, according  to  our  experience,  is  better  performed  and  with 
more  uniform  success,  if  a  few  milligrams  of  the  substance  are 
placed  on  a  porcelain  surface  together  with  one  or  two  drops  of 
NH4OH  and  evaporated  to  dryness  on  a  water-bath;  the  violet 
tinge  is  here  seen  against  the  white  surface.  In  trying  to  dissolve 
some  of  the  salt  in  50  per  cent  alcohol  it  was  discovered  that 
although  very  little  went  into  solution  the  latter  became  colored 
with  this  same  violet  tint.  This  pyrimidine  also  resembles  uric 
acid  in  two  reactions,  namely,  the  murexide  and  SchifFs  tests;  the 
murexide  test  is  given  very  brilliantly  indeed.  Addition  of  bro- 
mine water  to  an  aqueous  solution  was  found  to  produce  a  deep 
reddish-purple  color  which  vanished,  leaving  a  yellow  solution, 
when  the  bromine  was  in  excess.  The  NaN02-FeS04  reaction  is 
positive  if  the  triamino  pyrimidine  be  first  dissolved  in  boiling 
water;  this  is  probably  due  to  a  trace  of  the  diamino  being  formed 
by  the  action  of  the  water  as,  theoretically,  if  the  5  position  is 
occupied  by  an  amino  group  no  isonitroso  derivative  can  be  formed. 


N 


Calculated  for 
(C4H7N£0)H2S04+H20: 

27.24 


Found: 

27.87 


31  Traube:  Ber.  d.  deutsch.  chem.  Gesellsch.,  xxxiii,  pp.  1371-1383,  1900. 


Israel  S.  Kleiner 


463 


In  using  this  salt  in  physiological  experiments  we  again  obtained 
results  quite  different  from  those  reported  by  Steudel. 

Experiment  14.  February  11.  2:45  p.m.  Bitch  weighing  9.8  kg.  (same 
animal  as  in  Experiment  15)  was  given  1.58  grams  of  the  sulphate  of  this 
pyrimidine  mixed  with  about  180  grams  of  chopped  meat  and  some  bone 
meal.    No  unusual  symptoms  were  noticed  by  4:00  p.m. 

4:30;  5:00;  5:30;  7:40;  9:15  p.m.;  animal  observed  and  was  well  and  play- 
ful. 

February  12.  8:50  a.m.  Animal  well.  Urine,  134  cc;  dark  orange-red 
in  color;  specific  gravity,  1.049;  acid;  no  albumin.  Some  of  the  urine  was 
made  acid  with  H2S04  and  was  concentrated  to  small  volume.  HgS04 
solution  was  added  and  the  precipitate  filtered  off;  a  few  crystals  were  found 
and,  as  they  gave  a  violet  color  on  treatment  with  NH4OH  and  evaporation, 
were  probably  some  triamino-sulphate  which  had  crystallized  before  adding 
the  HgS04.  The  mercury  precipitate,  was  unfortunately  lost  through  an 
accident. 

4:00  p.m.    Fed  meat  and  bone. 

February  13.    a.m.    Urine  light  yellow  in  color. 

Relatively  larger  doses  were  fed  in  suspension  to  a  rabbit  and 
a  guinea  pig  with  similarly  negative  results;  these  are  summed  up 
in  the  following  table  (Table  V). 


table  v. 

Animal  Experiments:  2,  4,  5-triamino-6-oxypyrimidine. 


NUMBER  AND 
ANIMAL 


(14)  Dog. 


(10)  Guinea 

Pig 
(young). 


kg. 

9.8 


(13)  Rabbit..  2.5 


0.13 


AMOUNT  GIVEN 


Total 


gram 

1.58 


0.2 
0.5 


0.13 


Per 
kilo- 
gram 


MANNER  OF 
ADMINISTRATION 


REMARKS  AND  RESULTS 


gram 

0.16 


Per  os 


0.08  Per  os 
0.20 


1.0   !  Per  os 


No  toxic  effect.  Some 
excreted  (?).  Urine 
red. 

No  toxic  effect.  Sec- 
ond dose  four  days 
after  first.  Urine 
red  after  second 
dose. 

Fed,  suspended  in 
saccharose,  solu- 
tion, from  a  pip- 
ette. No  toxic 
effects.  Urine  col- 
ored dark  red. 


464 


Action  of  Certain  Pyrimidines 


It  is  accordingly  evident  that  even  the  triamino  compound, 
which  Steudel  claims  is  the  more  toxic  of  the  two,  has  no  harmful 
influence  upon  the  organism  when  administered  by  way  of  the 
mouth. 

Cyanacetylguanidine. 

HN— CO 

I  I 
HN  =  C  CH2 

I  I 
H2N  CN 

The  preparation  and  properties  of  cyanacetylguanidine  are 
described  above  in  the  description  of  the  process  of  making  2,4- 
diamino-6-oxypyrimidine. 

This  compound  was  used  because  it  is  a  precursor  of  the  diamino 
and  triamino  pyrimidines  just  described  and  might  readily  be  pres- 
ent as  an  impurity  if  these  compounds  were  carelessly  prepared.  In- 
asmuch as  from  the  following  experiments  it  is  seen  to  be  toxic 
after  injection,  a  reason  for  the  difference  between  our  results  and 
SteudeFs  is  thus  suggested. 

Experiment  28.  March  30.  12:30  m.  Injected  subcutaneously,  into 
guinea  pig  weighing  680  grams,  0.38  gram  cyanacetylguanidine  in  15  cc. 
water. 

4:00  p.m.  Animal  shows  hyperexcitability. 
6:20  p.m.  Still  very  excitable. 

March  31.  1:15  p.m.  Apparently  well  except  for  continued  hyperexcit- 
able  state,  which  is  not  as  great  as  on  the  previous  day. 

4:00  p.m.  Has  eaten  15  grams  oats  and  95  grams  carrots  during  twenty- 
four  hours.  Weight  668  grams.  Urine,  43  cc;  alkaline;  specific  gravity, 
1.031;  no  albumin  present;  strong  NaN02-FeS04  test;  upon  addition  of 
NaN02  in  substance,  and  H2S04  a  pink  isonitroso  derivative  was  obtained 
which  amounted  to  0.161  gram,  if  computed  to  total  volume.  This  was 
analyzed  with  the  following  results. 

Calculated  for 
C4H5N5O2  ( = isonitroso 

derivative  of 
cyanacetylguanidine) :  Found : 

N.   45.16  36.47 

5:10  p.m.    Apparently  well. 

April  1.  4:00  p.m.  Has  eaten  15  grams  oats  and  105  grams  carrots. 
Weight,  664  grams.  Urine,  46  cc. ;  alkaline;  specific  gravity,  1.030;  no  albu- 
min; NaN02-FeS04  test  positive. 


Israel  S.  Kleiner 


465 


April  2.  4:00  p.m.  Weight,  667  grams,  Urine,  38  cc;  NaN02-FeS04 
test  negative. 

Experiment  31.  April  2.  11:45  a.m.  Young  guinea  pig,  weighing  191 
grams,  given  0.4  gram  cyanacetylguanidine  in  17  cc.  water  by  subcutan- 
eous injection. 

12:15  m.   Apparently  well. 

2:10  p.m.  Animal  found  in  violent  spasms,  especially  the  posterior  parts 
of  the  body.    There  is  hyperexcitability. 

2:20  p.m.  Head  raised  a  little  more  and  pig  runs  around  some,  pawing 
at  its  chin  at  intervals.    Twitchings  continue. 

4 :13  p.m.  Violent  convulsion;  lies  on  its  side  and  moves  its  limbs  rapidly. 

4:18  p.m.  Animal  gradually  rights  itself  and  grips  the  side  of  the  wire 
cage  with  its  teeth.  Waves  of  convulsions,  starting  at  the  posterior  part 
and  running  forward,  occur. 

4:22  p.m.  Dies  in  the  same  position;  body  quickly  in  rigor.  The  urine 
excreted,  2  cc,  was  found  to  contain  no  albumin  but  on  addition  of  NaN02 
and  H2SO4  a  pink  precipitate  appeared  which  after  dissolving  in  Na2CC>3 
and  adding  FeS04  produced  the  deep  prussian  blue  color. 

Experiment  30.  March  SI.  2:55  p.m.  A  dog  weighing  5.8  kg.  was  fed 
100  grams  chopped  meat  containing  0.94  gram  cyanacetylguanidine. 

3:10  to  3:20;  4:25  to  4:30  p.m.  Apparently  no  effects. 

4:50  p.m.  Drank  water;  no  nausea. 

April  1.  9:15  a.m.  Dog  apparently  well.  3:00  p.m.  Fed  meat,  lard,  bone 
and  cracker  meal.  Urine,  226  cc;  acid;  specific  gravity,  1.025;  no  albumin; 
strong  NaN02-FeS04  test.  To  an  aliquot  portion  was  added  solid  NaN02 
and  H2SO4  and  the  reddish  brown  precipitate  which  amounted  to  0.389  gram 
analyzed. 


April  2.  3:00  p.m.  Dog  well.  Urine,  138  cc;  specific  gravity,  1.032; 
acid;  no  albumin;  strong  NaNCVFeSC^  test.  No  loss  of  appelite  or  other 
unfavorable  symptoms. 

April  3.  9:30  a.m.  Dog  well.  Weight,  5.6  kg.  Urine  gives  uncertain 
NaN02-FeS04  test. 

These  and  one  other  experiment  are  summarized  in  Table  VI. 
The  low  nitrogen  values  found  in  Experiments  28  and  30  suggest 
the  possibility  of  a  deaminization  of  cyanacetylguanidine  in  the 
body.  The  substitution  of  O  for  NH  in  its  isonitroso  derivative 
would  result  in  a  compound  containing  35.90  per  cent  of  nitrogen; 
the  figures  found,  36.47  per  cent  and  34.43  per  cent,  correspond 
with  this  percentage. 


Calculated  for 
C4H5N5O2  (  =  isonitroso 

derivative  of 
cyanacetylguanidine) : 

  45.16 


N 


Found: 

34.43 


466  Action  of  Certain  Pyrimidines 

TABLE  VI. 


Animal  Experiments:  Cyanacetylguanidine. 


NUMBER  AND 

AMOUNT  GIVEN 

MANNER  OP 

WEIGHT 

ANIMAL 

Total 

Per 
kilo- 

gram 

ADMINISTRATION 

REMARKS  AND  RESULTS 

kg. 

gram 

gram 

(28)  Guinea 

Hyperexcitability . 

pig.... 

0.68 

0.38 

0.56 

Subcutaneously 

Excreted  consider- 
able as  a  deamin- 
ized  (?)  substance. 

(31)  Guinea 

Hyperexcitability. 

pig.... 

0.19 

0.40 

2.1 

Subcutaneously 

Violent  convul- 
sions. Fatal  in 
four  and  three 
quarters  hours. 

(29)  Dog  

8.4 

0.70 

0.08 

Per  os 

No  symptoms. Urine 
gave  positive  Na- 
N02-FeS04  test. 
No  albuminuria. 

(30)  Dog  

5.8 

0.94 

0.16 

Per  os 

No  harmful  effect. 
Considerable  ex- 
creted asadeamin- 
ized(?)  substance. 

This  toxic  action  agrees  with  the  results'  of  some  unpublished 
trials  by  Mr.  J.  J.  Costello,  who  observed  similar  effects  in  Pro- 
fessor Mendel's  laboratory  when  the  sulphate  of  this  compound 
was  subcutaneouly  injected.    A  few  of  his  figures  follow. 

Dose  per  kilogram 


of  guinea  pig.  Results 

0 . 92  Hyperexcitability-recovery. 

0.96  Hyperexcitability  for  two  days-recovery. 

0.96  Death  in  sixteen  hours. 

1.02  Death  in  fourteen  hours. 

2.26  Death  in  three  and  one-half  hours. 


DISCUSSION. 

In  considering  the  physiological  and  pharmacological  behavior 
of  the  members  of  this  series  the  most  striking  fact  is  the  toxicity 
of  5-aminomalonyl  guanidine  with  its  chief  effect  upon  the  epithe- 
lium of  the  convoluted  tubules.    Its  harmlessness  when  adminis- 


Israel  S.  Kleiner 


467 


tered  per  os  may  be  due  either  to  an  absorption  so  slow  as  to  allow 
of  elimination  before  a  toxic  concentration  is  reached,  or  to  a  trans- 
formation— perhaps  by  deaminization — into  a  non-toxic  compound 
in  the  intestinal  wall.  The  toxicity  after  subcutaneous  adminis- 
tration may  possibly  be  attributable  to  some  hydrolytic  or  oxida- 
tion product  formed  during  solution  inasmuch  as  the  solution 
quickly  assumes  a  red  color. 

The  absence  of  hypnotic  powers  in  barbituric  acid  and  malonyl- 
guanidine  is  in  harmony  with  the  ineffectiveness  of  the  lower  alkyl 
barbituric  acid  derivatives  and  of  5,5-dipropylmalonylguanidine.32 
The  diarrheal  action  of  barbituric  acid  is  noteworthy  because  of 
a  similar  action  ascribed  to  alloxan.33 

Steudel's34  claim  that  2,4-diamino-6-oxypyrimidine  and  2,4,5- 
triamino-6-oxypyrimidine  are  toxic,  cannot  be  substantiated.  In 
duplicating  his  experiments  in  which  he  fed  these  compounds  to  a 
dog,  no  similar  results  could  be  obtained;  the  animal  used  was  a 
very  playful  one  as  was  SteudeFs  but  it  did  not  become  less  lively 
after  ingesting  these  substances,  nor  was  vomiting  or  albuminuria 
observed  or  any  other  of  the  effects  noted  by  that  author.  The 
lethal  doses  for  rats  he  gives  as  0.2  gram  and  0.1  gram  for  the  sul- 
phates of  the  diamino  and  triamino  compounds,  respectively,  when 
injected  subcutaneoulsy.  The  smallest  volumes  which  can  possi- 
bly contain  these  amounts  at  43°  are  40  cc.  and  20  cc.  respectively. 
Moreover,  it  was  shown  above  that  such  concentrations  are  not 
suitable  for  injection  and  this  leads  us  to  believe  that  Steudel  used 
products  which  were  more  soluble  than  these  aminopyrimidines. 
Moreover  he  published  no  analyses  of  his  compounds.  Cyanace- 
tylguanidine,  however,  is  a  precursor  of  both  pyrimidines;  it  is 
quite  soluble  as  is  also  its  sulphate;  and  finally,  when  injected  sub- 
cut  aneously  it  is  toxic.  These  properties  would  indicate  that  this 
compound  was  an  admixture  of  Steudel's  preparations  and  would 
account  for  their  toxic  action.  However,  when  fed  to  dogs,  cyana- 
cetylguanidine  is  not  toxic  although  his  preparations  were;  and 
the  only  apparent  explanation  for  this  is  that  still  another  com- 
taminating  substance  was  responsible  in  this  case.  That  cyana- 
cetylguanidine  is  toxic  is  not  surprising  since,  from  its  structure, 

32  Fischer  and  von  Mering:  Therapie  der  Gegenwart,  v,  pp.  97-101,  1903. 

33  Koehne:  Inaugural  Dissertation,  Rostock,  1894,  40  pp. 
"Steudel:  Zeitschr.  f.  physiol.  Chem.,  xxxii,  pp.  285-290,  1901. 


468 


Action  of  Certain  Pyrimidines 


HN— CO 

I  I 
HNC  CH2 

I  I 
H2N  CN 

it  might  possess  the  properties  of  guanidine  or  of  nit  riles.  Guani- 
dine,  the  toxicity  of  which'  has  long  been  known,  causes35  peculiar 
shaking  movements  of  the  head  and  ears,  paralysis  of  the  hind 
limbs,  clonic  muscular  contractions  and  muscular  twitchings  of  the 
entire  body.  Different  nitriles  have  different  effects  but  the  typi- 
cal phenomena  are  described36  as  vomiting,  dyspnoea,  tetanic  con- 
vulsions and  opisthotonus.  Hence,  probably  cyanacetylguanidine 
embraces  some  of  the  toxic  effects  of  both  of  these  poisons  (see 
Experiments  28  and  31). 

The  behavior  of  2,4-diamino-6-oxypyrimidine  and  cyanacetyl- 
guanidine in  the  body  affords  suggestions  for  further  work  upon  the 
intermediary  metabolism  of  these  substances,  as  the  few  experi- 
ments indicate  that  a  deaminization  may  occur  in  vivo.  The  dif- 
ferences between  the  theoretical  percentage  of  N  for  the  compounds 
administered  and  those  recovered  from  the  urine  are  too  great 
(6  to  11  per  cent)  to  be  ascribed  to  the  method  of  analysis  or  to 
faulty  technique.  Moreover,  an  analysis  of  the  pure  isonitroso 
derivative  of  the  diamino  pyrimidine  by  the  same  method  gave  a 
satisfactory  nitrogen  value.  The  possibilities  for  the  transforma- 
tion of  this  pyrimidine  are  shown  by  the  following  scheme. 

HN— CO  HN— CO  HN— CO 

II  .11  II 

H2NC    CH     +  H20  =  NH3  +  OC    CH2    or  HNC  CH2 

II     II  II  II 

N— CNH2  HN— CNH         HN— CO 

With  cyanacetylguanidine  a  somewhat  similar  problem  is  pre- 
sented as  deaminization  can  result  in  one  of  three  compounds: 

HN— CO  HN— CO       HN— CO  HN— CO 

II  lilt  II 

HNC    CH2  +  H20  =  NH3  +  OC    CH2  or  OC    CH2  or  HN— C  CH2 

II  I      I  I      I  II 

H2N    CN  H2N    CN       HN— CNH  HN— CO 

35  Gergens  and  Baumann:  Arch.  f.  d.  ges.  Physiol.,  xii,  pp.  205-214,  1876; 
Pommerenig :  Beitr.  z.  chem.  Physiol,  u.  Path.,  i,  pp.  561-566,  1901. 
86  Kobert :  Lehrbuch  der  Intoxikationen,  Stuttgart,  ii,  p.  862,  1906. 


Israel  S.  Kleiner 


469 


If  either  of  the  last  two  complexes  result  it  is  of  great  interest 
as  no  precisely  similar  transformation  of  an  acyclic  into  a  cyclic 
compound  is  known  in  physiology. 

HN— 

I 

Lusini's  conclusion  that  the  grouping  OC    has  first  a  stimulat- 

I 

HN— 

ing  and  then  an  inhibiting  action  on  the  nerve  centers  and  that 
HN— CO 

the  grouping  1     has  no  such  power  can  not  be  substan- 

I 

tiated  inasmuch  as  barbituric  acid,  which  is  non-toxic,  contains 
this  urea  grouping  and  differs  very  little  in  structure  from  alloxan 
which  Lusini  found  to  be  toxic. 

SUMMARY. 

The  administration  of  barbituric  acid  per  os  is  followed  by  no 
marked  physiological  effects  except  diarrhea;  when  given  subcu- 
taneously  the  free  pyrimidine  has  a  local  action  on  the  tissues  due 
to  its  acid  properties.    The  sodium  salt  has  no  local  action. 

Malonyl  guanidine  when  fed,  or  when  injected  subcutaneously 
as  the  sodium  salt,  provokes  no  noteworthy  symptoms.  5-Amino- 
malonylguanidine  hydrochloride,  2, 4-diamino-6-oxypyrimidine  sul- 
phate and  2,4,5-triamino-6-oxypyrimidine  sulphate,  when  fed,  are 
also  without  marked  action. 

Subcutaneous  injection  of  5-aminomalonylguanidine  hydro- 
chloride leads  to  grave  changes  in  the  tubular  epithelium  of  the 
kidney;  casts  and  albumin  abound  in  the  urine;  and  death  fre- 
quently results. 

2,4-Diamino-6-oxypyrimidine  sulphate  and  2,4,51triamino-6-oxy- 
pyrimidine  sulphate,  which  Steudel  reported  as  toxic,  are  too  insol- 
uble to  inject  in  appreciable  quantity.  Inasmuch  as  cyanacetyl- 
guanidine,  a  precursor  of  both  of  these,  is  quite  soluble,  and  was 
found  to  be  toxic  when  injected  subcutaneously,  doubt  is  expressed 
as  to  the  purity  of  the  diamino  and  triamino  pyrimidines  used  by 
Steudel,  especially  as  this  author  also  observed  nausea,  etc.,  after 
feeding  them  to  dogs,  whereas  no  symptoms  whatever  occurred 
in  the  present  investigation  under  similar  conditions. 


47o 


Action  of  Certain  Pyrimidines 


A  color  reaction  is  described  which  is  common  to  all  of  this  series, 
although  2,4,5-triamino-6-oxypyrimidine  and  5-aminomalonylgua- 
nidine  do  not  react  well.  By  aid  of  this  reaction  and  in  other  ways, 
evidence  was  gained  that,  after  administration  of  a  compound  of 
this  series  there  was  excreted  in  the  urine  the  compound  used  (or 
a  derivative)  in  every  case  except  with  5-aminomalonylguanidine, 
and  perhaps  2,4,5-triamino-6-oxypyrimidine. 

Evidence  is  presented  to  indicate  that  2,4-diamino-6-oxypy- 
rimidine  and  cyanacetylguanidine  may  be  deaminized  in  the  body. 

'  My  thanks  are  due  Prof.  Lafayette  B.  Mendel  who  directed  the 
physiological  investigations  and  Prof.  Treat  B.  Johnson,  who  aided 
and  advised  in  the  syntheses  of  the  compounds  employed  as  well 
as  in  the  questions  of  organic  chemistry  involved. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  XII,  No.  1,  1912. 


THE  INFLUENCE  OF  SODIUM  TARTRATE  UPON  THE 
ELIMINATION   OF  CERTAIN  URINARY  CONSTITU- 
ENTS DURING  PHLORHIZIN  DIABETES. 

By  FRANK  P.  UNDERHILL. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
.   New  Haven,  Connecticut.) 

(Received  for  publication,  May  25,  1912.) 

In  recent  communications  by  Baer  and  Blum1  it  is  pointed  out 
that  the  subcutaneous  administration  of  a  series  of  organic  com- 
pounds containing  two  carboxyl  groups  exercises  a  remarkable 
inhibitory  influence  upon  the  elimination  of  urinary  nitrogen  and 
dextrose  in  dogs  with  phlorhizin  diabetes.  Among  the  substances 
possessing  this  property  may  be  mentioned  glutaric  and  tartaric 
acids  and  their  salts. 

The  results  obtained  by  these  authors  are  so  striking  and  of  such 
fundamental  importance  in  the  interpretation  of  the  mechanism 
of  phlorhizin  diabetes  that  a  reinvestigation  of  the  problem  seemed 
desirable.  Accordingly  experiments  have  been  planned  similar 
to  those  of  Baer  and  Blum  the  details  of  which  are  appended.2 
The  investigation  has  corroborated  the  reported  results  but  has 
yielded  an  explanation  for  the  phenomena  observed  which  is  differ- 
ent from  that  put  forth  by  Baer  and  Blum.  Our  work  has  been 
confined  to  the  study  of  the  action  of  a  single  compound  used  by 
Baer  and  Blum,  namely,  sodium  tartrate,  prepared  by  neutrali- 
zation of  the  racemic  crystalline  tartaric  acid  (Kahlbaum  and  other 
preparations)  with  sodium  carbonate.  Both  rabbits  and  dogs  were 
employed  as  experimental  animals. 

*Baer  and  Blum:  Hofmeister's  Beitrdge,  x,  p.  80,  1907;  xi,  p.  102,  1908; 
Arch.  f.  exp.  Path.  u.  Pharm.,  lxv,  p.  1,  1911. 

2  A  notice  of  this  investigation  was  communicated  to  the  Society  for  Ex- 
perimental Biology  and  Medicine,  May  15,  1912. 

ii5 


1 1 6      Sodium  Tartrate  and  Phlorhizin  Diabetes 


After  the  completion  of  our  experiments  a  preliminary  account 
of  the  influence  of  glutaric  acid  on  phlorhizin  diabetes  was  reported 
by  A.  I.  Ringer.3  In  this  communication  Ringer  has  entirely  failed 
to  confirm  the  reported  results  of  Baer  and  Blum  with  respect  to 
glutaric  acid. 

Methods.  The  general  plan  of  experimentation  was  similar  to 
that  of  Baer  and  Blum.  Phlorhizin  diabetes  was  established  for 
a  preliminary  period  (usually  three  days)  in  the  fasting  animal, 
the  drug  being  given  subcutaneously  once  daily  in  sodium  carbonate 
solution.  Water  was  allowed  ad  libitum.  Urine  was  collected  in 
twenty-four-hour  periods  either  by  compression  of  the  bladder  in 
rabbits  or  by  catheterization  in  the  case  of  dogs.  Tartrate  admin- 
istration occurred  immediately  after  the  phlorhizin  injection  and  the 
quantities  of  tartaric  acid  specified  in  the  tables  were  subcutane- 
ously injected  subsequent  to  neutralization  with  sodium  carbonate. 

In  our  preliminary  trials  we  repeated  the  work  of  Baer  and  Blum 
employing  rabbits  instead  of  dogs.  As  may  be  seen  from  tables 
1,  2,  3,  and  4,  sodium  tartrate  administered  subcutaneously  to 
rabbits  with  phlorhizin  diabetes  promptly  causes  a  very  decided 
diminution  in  the  output  of  total  nitrogen  and  dextrose.  It  will 
also  be  observed,  however,  that  urine  secretion  is  greatly  diminished 
and  in  some  of  the  experiments,  of  which  the  appended  are  a  few 
examples  only,  was  completely  inhibited.  From  the  data  in  the 
first  four  tables  it  is  evident  that  suppression  of  urine  is  sufficient 
to  account  for  the  very  great  decrease  in  the  output  of  the  urinary  con- 
stituents under  consideration.  When  the  urine  secretion  was  not 
entirely  inhibited  we  have  at  times  obtained  water-clear  twenty- 
four-hour  specimens  of  fair  volume  in  which  no  trace  of  nitrogen  or 
dextrose  could  be  detected. 

Experiments  with  dogs  yielded  results  in  accord  with  those 
obtained  with  rabbits  as  may  be  seen  from  the  examples  cited  in 
tables  5  and  6.  In  experiment  7,  dog  1,  table  5,  the  change  in 
the  output  of  urine  and  of  the  urinary  constituents  under  discussion 
shows  a  striking  similarity  to  that  observed  in  rabbits.  Experi- 
ment 8,  dog  3,  table  6,  is  inserted  to  show  that  at  times  one  dog  may 
perhaps  be  less  susceptible  to  the  action  of  tartrate  than  other  indi- 
viduals, and  that  water  may  be  eliminated  by  the  kidney  even 

3  Ringer:  Proceedings  of  the  Society  for  Experimental  Biology  and  Medicine, 
ix,  p.  54,  1912. 


Frank  P.  Underhill  117 

TABLE  1. 
EXPERIMENT  1,   RABBIT  A. 


Male  rabbit  of  2200  grams  received  daily  subcutaneous  injection  of  0.25  gram 

phtorhizin. 


DATE 
1911 

Volume 

UR 

Specific 
gravity 

INE 



Total 
Nitrogen 

Dextrose 

REMARKS 

November 

cc. 

grams 

grams 

14 

90 

1 .040 

1 .85 

2.72 

15 

100 

1.026 

1.60 

1.80 

16 

100 

1.030 

1.99 

1.43 

17 

30 

OK) 

o  13 

u .  uu 

OUUCU  IHIltJO  Uo    1I1J  cl  LlOIl 

. 

of   3  0    crvnmc;  f.nrf.Qrir* 

clUlLl,  lie U  tl  d>l±£i*5\JL  WJ.LI1 

Na2C03,    in    40  cc. 

water. 

18 

8 

0.00 

0.00 

Animal    lies    in  deep 

coma.    Does  not  re- 

spond to  stimulation. 

19 

0 

Heart  beat  and  respi- 

ration very  slow. 

20 

0 

Animal  found  dead. 

Bladder  empty.  No 

urine  secreted  for  24 

hours. 

when  the  latter  is  no  longer  in  a  condition  to  normally  secrete  the 
organic  constituents  of  the  urine.  With  rabbits  exactly  analogous 
conditions  may  obtain.  In  other  words,  a  dose  of  sodium  tartrate 
which  in  the  majority  of  rabbits  or  dogs  causes  suppression  of  urine 
may  exert  only  a  slight  influence  in  this  direction  in  a  small  number 
of  individuals.  An  inspection  of  the  data  presented  by  Baer  and 
Blum  points  to  the  same  conclusion,  and  it  is  possible  that  the  .nega- 
tive results  reported  by  Ringer  may  be  due  to  the  same  fact.  This 
seems  hardly  likely,  however,  and  as  a  possible  explanation  of 
Ringer's  failure  to  corroborate  the  findings  of  Baer  and  Blum  we 
would  call  attention  to  the  fact  that  Ringer  administered  his  glutaric 
acid  solutions  in  three  equal  doses  during  the  course  of  the  day  and 
in  this  way  failed,  perhaps,  to  overwhelm  the  capacity  of  the  animal 
to  transform  the  compound  into  a  harmless  derivative. 


1 1 8      Sodium  Tartrate  and  Phlorhizin  Diabetes 


TABLE  2. 
EXPERIMENT  2,  RABBIT  B. 

Female  rabbit  of  2300  grams  received  daily  subcutaneous  injection  of  0.25 

gram  phlorhizin. 


DATE 
1911 

URINE 

REMARKS 

Volume 

Specific 
gravity 

Total 
nitrogen 

Dextrose 

November 

cc. 

grams 

grams 

14 

115 

1.030 

1.66 

3.58 

15 

75 

1.036 

1.58 

1.39 

16 

100 

1.030 

1.82 

1.28 

17 

55 

0.06 

0.17 

Ssi  l  r*pii  t  €k  n  pah  c    iniopf  ir\M 
OUUtliliallCUUS    1I1J  CO  UlUIl 

r»f  3  0   D"Tfimcj  tnrf  nrir 

npifl    npiitrn  1  1 7Pfl  wif.Vi 

cliV-'lVl,    11C  Li  Ul  CLlHiCU.  Willi 

Na2C03,    in    40  cc. 

water. 

18 

10 

0.05 

0.00 

19 

0 

Animal  lies  in  cage,  can- 

not  stand.    Has  no 

muscular  control. 

Heart  beat  and  respi- 

ration are  greatly  ac- 

celerated. 

20 

0 

Found  dead.    No  urine 

secreted  for  twenty- 

four  hours. 

From  the  data  in  tables  1,  2,  3,  and  4,  it  is  evident  that  sodium 
tartrate  produces  its  unique  influence  upon  the  elimination  of  urinary 
nitrogen  and  dextrose  in  phlorhizinized  animals  by  causing  a  partial 
or  complete  suppression  of  urine.  In  order  to  determine  further 
the  correctness  of  this  conclusion,  sections  of  the  kidneys  were 
preserved  and  sent  to  Prof.  H.  Gideon  Wells  of  the  University  of 
Chicago  to  whom  I  am  greatly  indebted  for  the  examination  of  the 
tissues.  In  his  report  Professor  Wells  says  in  part,  "The  greater 
part  of  the  epithelium  of  the  convoluted  tubules  is  entirely  necrotic, 
and  most  of  the  tubules,  almost  all,  in  fact,  are  occluded  by  large 
hyaline  and  granular  casts,  frequently  containing  more  or  less 
hemoglobin.  It  is  easy  to  understand  that  such  a  kidney  could 
not  secrete.  It  is  quite  as  severe  a  change  as  I  have  ever  seen  in 
experimental  nephritis.    There  is  very  little  difference  between  any 


Frank  P.  Underhill 


119 


TABLE  3. 
EXPERIMENT  3,  RABBIT  C. 

Female  rabbit  of  2200  grams  received  daily  subcutaneous  injection  of  0.25 

gram  phlorhizin. 


URINE 

DATE 
1911 

Volume 

Specific 
gravity 

Total 
nitrogen 

Dextrose 

REMARKS 

November 

cc. 

grams 

grams 

21 

85 

1.026 

0.95 

1.88 

Drank  80  cc.  water. 

22 

75 

1.030 

1.30 

1.16 

Drank  45  cc.  water. 

23 

75 

1.030 

1.18 

0.87 

Drank  50  cc.  water. 

24 

30 

1.012 

0.00 

0.00 

Subcutaneous  injection 
of  1.75  grams  tartaric 
acid,  neutralized  with 
Na2C03,  in  30  cc. 
water.  Drank  140  cc. 
water. 

25 

40 

1.015 

0.07 

0.00 

Would  not  drink.  Par- 
tial prolapse  of  uterus. 
Animal  killed.  On 
autopsy  all  organs 
appeared  normal  ex- 
cept    the  kidneys 

• 

which  seemed  very 
pale  and  soft. 

of  the  specimens,  and  that  only  in  degree.  The  glomerules  show 
almost  no  change  beyond  an  occasional  small  hemorrhage." 
Concerning  the  histology  of  the  dog  kidneys  Professor  Wells 
reported  that  vacuolization  was  much  more  prominent  than 
necrosis. 

The  histological  picture  therefore  coincides  with  the  other  data. 
In  their  investigation  Baer  and  Blum  apparently  made  no  histolo- 
gical study  of  the  kidneys,  hence  their  failure  to  fully  recognize 
the  cause  of  the  diminished  excretion  of  the  urinary  constituents. 
It  is  only  fair,  however,  to  add  that  the  suspicion  of  a  kidney 
factor  must  have  entered  into  their  ideas  since  they  have  recorded 
some  experiments  with  this  point  in  mind.4  But  they  tested  the 
secretory  power  of  the  kidney  by  means  of  inorganic  salts  only  and 

4  Baer  and  Blum:  Arch.  f.  exp.  Path.  u.  Pharm.,  lxv,  p.  1,  1911. 


i2o      Sodium  Tartrate  and  Phlorhizin  Diabetes 


TABLE  4. 
EXPERIMENT  4,   RABBIT  D. 


Female  rabbit  of  2200  grams  received  daily  subcutaneous  injection  of  0.25 
gram  phlorhizin. 




DATE 

1911 



Volume 

URINE 

Specific  Total 
gravity  nitrogen 

Dextrose 

REMARKS 

November 

cc. 

grams 

grams 

 .  

21 

125 

1.024 

1.12 

2.75 

Drank  70  cc.  water. 

22 

GO 

1.036 

1.17 

1.28 

Drank  25  cc.  water. 

23 

50 

1.040 

0.92 

1.19 

Drank  45  cc.  water. 

24 

18 

0.05 

0.00 

Subcutaneous  injection 

of  2.0  gram  tartaric 

acid,  neutralized  with 

Na2C03,    in    40  cc. 

water.    Animal  drank 

125  cc.  water. 

25 

Ml 

2  drops 

n  on 

Tivitip  /*Mr\"fc   in    i  ckl  1  \i  liL-o 
Millie  tlUlo  111  Jell  J  -Hive 

mass.     Would  not 

drink. 

26 

3 

0.00 

Would  not  drink. 

27 

0 

At  5  p.m.  animal  was 

seized   with  convul- 

sions and  died  id  few 

moments.    At  autop- 

sy all  organs  appeared 

normal  except  the  kid- 

neys which  were  pale 

and  soft. 

report  no  change  in  the  elimination  of  these  compounds  and  there- 
fore conclude  that  kidney  secretory  factors  are  not  primarily  account- 
able for  their  results.  However,  it  does  not  necessarily  follow- 
that  in  a  given  form  of  nephritis  inorganic  salts  alone  may  not 
be  eliminated,  nor  is  it  fair  to  assume  that,  because  one  substance 
may  be  excreted,  a  second  compound  of  an  entirely  different  chemi- 
cal nature  will  behave  in  the  same  manner.  If  these  results  of 
Baer  and  Blum  with  inorganic  salts  are  accepted  our  results  indicate 
the  correctness  of  our  contention.  It  is  not  our  intention,  however, 
at  this  time  to  enter  more  deeply  into  the  conditions  attendant 
upon  tartrate  nephritis  but  rather  to  indicate  that  a  nephritic 


# 


Frank  P.  Underhill 


I  2  I 


TABLE  5. 
EXPERIMENT  7,   DOG  1. 


Full-grown  bitch  of  9  kilos  received  daily  subcutaneous  injection  of  1.5  grams 

phlorhizin. 


tJRINE 

DATE 
1912 

REMARKS 

Volume 

Specific 
gravity 

Total 
nitrogen 

Dextrose 

February 

cc. 

grama 

grams 

6 

220 

1.070 

6.42 

32.82 

D  :  N  ratio  =  5.11. 
Animal  drank  120  cc. 
water. 

7 

415 

1.074 

11.70 

43.33 

D  :  N  ratio  =  3.70. 
Animal  drank  250  cc. 
water. 

8 

600 

1.053 

11.98 

40.06 

D  :  N  ratio  =  3.34. 
Animal   drank   70  cc. 
water. 

9 

125 

1.008 

0.16 

0.00 

8.0  grams  tartaric  acid, 
neutralized  with  Na2- 
C03,  were  subcutane- 
ously   injected,  dis- 
solved in  50  cc.  water. 

10 

10 

_ 

0.075 

0.25 

Animal  has  lost  control 

• 

of  muscles   and  lies 
in  cage.    Vomits  any 
water  given.    At  the 
close  of  this  day  it 
was    apparent  that 
dog  would  not  survive 
the    night.  Animal 
was  killed  by  chloro- 
form.   All  organs  ap- 
peared normal. 

condicion  must  be  taken  into  consideration  in  the  discussion  of  the 
results  reported  by  Baer  and  Blum .  Some  of  the  factors  of  tartrate 
nephritis  will  be  detailed  in  a  subsequent  communication  shortly  to 
appear,  the  work  of  which  has  already  been  completed. 

The  present  problem  has  also  been  attacked  from  another  stand- 
point. It  is  quite  conceivable  that  phlorhizin,  acting,  presumably, 
specifically  upon  the  kidney  structure,  may  render  the  latter  unus- 
ually sensitive  to  tartrate  action  and  therefore  that  the  combina- 


122      Sodium  Tartrate  and  Phlorhizin  Diabetes 


TABLE  6. 
EXPERIMENT  8,  DOG  3. 


Full-grown  bitch  of  11.0  kilos  received  daily  subcutaneous  injection  of  1.5 
grams  phlorhizin. 


URINE 

DATE 

1912 

Volume 

Specific 
gravity 

Total 
Nitrogen 

Dextrose 

REMARKS 

March 

cc. 

grams 

grams 

5 

240 

1.070+ 

4.44 

31.05 

D  :  N  ratio  =  6.90. 
Animal  drank  370  cc. 
water. 

6 

240 

1.070+ 

8.11 

29.44 

D  :  N  ratio  =  3.63. 
Animal  drank  250  cc. 
water. 

t 

1.060 

8.79 

zy .  /  o 

D  :  JN  ratio  =  3.30. 

A        *            11             1  AAA 

Animal  drank  290  cc. 
water. 

8 

442 

1.035 

3.05 

12.88 

Subcutaneous  injection 
of  10.0  grams  tartaric 
acid,  dissolved  in  50 
cc.  fluid  and  neu- 
tralized with  Na2C03. 

9 

335 

1  .  uou 

7  fiS 

15.12 

After  injection  ani- 
mal vomited  repeat- 
edly. Could  not  drink 
because  of  vomiting. 

10 

209 

1.045 

1.27 

4.06 

11 

240 

1.016 

1.08 

1.02 

Animal  developed  ab- 
cess  at  site  of  injec- 
tion. In  a  weak  con- 
dition. Vomits  con- 
tinually. Killed  with 
chloroform.  At  au- 
topsy all  organs  ap- 
peared normal. 

tion  of  the  two  drugs  may  bring  about  changes  in  the  kidney  that 
neither  alone  could  accomplish.  If,  however,  sodium  tartrate  has 
a  specific  action  upon  kidney  secretion  this  should  be  manifested 
by  exclusion  of  the  phlorhizin  effect,  all  other  conditions  remaining 
unchanged.  We  have  endeavored  to  compass  this  result  and  in 
order  to  measure  the  extent  of  kidney  secretion  (in  the  absence  of 


Frank  P.  Underhill  123 


TABLE  7. 

EXPERIMENT  5,  RABBIT  E.  (Control). 

Male  rabbit  of  2%00  grams.    No  phlorhizin  was  given  throughout  experiment. 


URINE 

DATE 
1911 

Total 
nitrogen 

Creat- 
inine 

Creatine 

REMARKS 

November 

cc. 

grams 

milli- 
grams 

milli- 
grams 

22 

105 

1.012 

0.85 

• 

84 

9 

Animal  drank  150  cc. 
water. 

23 

65 

1.022 

n  si 

01 

24 

70 

1.020 

1.22 

78 

63 

Animal  drank  25  cc. 
water. 

25 

30 

1.015 

O  19 

Trace, 
too 
small 
to  es- 
timate 

Trace, 
too 
small 
to  es- 
timate 

QUUO  ULctilCUUb       JUJLJ  cl;  L1UH 

of  3.0  grams  tartaric 
acid,  neutralized  with 
Na2C03,  in  40  cc. 
water.  Animal  drank 
30  cc.  water.  No  symp- 
toms followed  injec- 
tion . 

26 

less 
than 
1  cc. 

- 

Animal  drank  110  cc. 
water. 

27 

0 

— 

— 

Animal    drank    60  cc. 
water.    Animal  ap- 
pears   normal  except 
that  head  is  rotated  to 
to  the  right.  During 
morning  had  one  con- 
vulsion. Recovered. 

• 

Found  dead.  At  autop- 
sy peritoneal  cavity 
contained  30  cc.  of 
clear  fluid  that  readily 
clotted.  The  kidneys 
presented  an  injected 
appearance.  All  other 
organs  seemed  normal. 
Bladder  contained  no 
urine. 

124      Sodium  Tartrate  and  Phlorhizin  Diabetes 

TABLE  8. 

experiment  6,  rabbit  f.  (Control). 


Female  rabbit  of  2200  grams.    No  phlorhizin  was  given  throughout  experiment. 


URINE 

DATE 

REMARKS 

1911 

Volume 

Specific 
gravity 

Total 
nitrogen 

Creat- 
inine 

Creatine 

November 

cc. 

grams 

milli- 
grams 

milli- 
grams 

22 

115 

1.011 

0.74 

90 

21 

Animal  drank  90  cc. 
water. 

23 

70 

1.020 

0.97 

111 

39 

Animal  drank  no  water. 

24 

60 

1.025 

0.90 

84 

48 

Animal  drank  50  cc. 
water. 

25 

32 

1.015 

0.15 

Trace, 

but 

too 
small 
to  es- 
timate 

Trace, 

but 

too 
small 
to  es- 
timate 

Subcutaneous  injection 
of  3.0  grams  tartaric 
acid,  neutralized  with 
Na2 C03,  in  40  cc.  water. 
No  symptoms  followed 
injection.  Animal 
drank  60  cc.  water. 

less 

Animal  drank  25  cc. 

26 

than 
5  cc. 

water.  Peculiar  posi- 
tion of  head  similar  to 
that  of  Rabbit  E. 

27 

0 

Animal  in  light  coma. 
Killed  with  chloro- 
form. Kidneys  were 
very  pale  and  soft. 
All  other  organs  were 
apparently  normal. 
Bladder  was  empty. 

glycosuria),  have  noted  the  excretion  of  urine  and  total  nitrogen  as 
usual  and,  in  addition,  the  elimination  of  creatinine  and  creatine, 
the  latter  compound  being  constantly  present  in  the  urine  of  our 
fasting  animals.  In  the  data  presented  in  tables  7  and  8  the  water 
intake  was  observed  in  order  to  discover  whether  diminished  volume 
of  urine  could  be  accounted  for  by  lack  of  water  consumption. 
It  will  be  seen  that  there  is  no  strict  correlation  between  the  intake 
and  the  output  of  water  in  the  tables  mentioned,  a  fact  which  also 
applies  to  the  data  contained  in  all  the  other  tables.    From  the 


Frank  P.  Underhill 


experiments  with  fasting  rabbits,  given  subcutaneous  injections 
of  tartrate  only,  it  is  evident  that  the  result  presented  is  one  induced 
specifically  by  the  tartrate  and  apparently  bears  little  or  no  relation 
to  the  application  of  phlorhizin. 

The  histological  examination  revealed  no  recognizable  differ- 
ences in  the  kidney  changes  of  specimens  taken  from  animals 
receiving  both  phlorhizin  and  tartrate  and  from  those  to  whom  only 
tartrate  had  been  administered.  In  his  partial  report  above,  Pro- 
fessor Wells  says,  ' 4  There  is  very  little  difference  between  any  of 
the  specimens,  and  that  only  in  degree. "  The  kidneys  taken  from 
animals  represented  in  tables  7  and  8  were  sent  to  Professor  Wells 
mixed  in  the  lot  excised  from  animals  having  had  an  injection  of 
both  phlorhizin  and  tartrate.  From  these  observations  it  is  appar- 
ent that  sodium  tartrate  alone  is  capable  of  inducing  a  particularly 
severe  form  of  nephritis  when  subcutaneously  introduced  into  rab- 
bits and  dogs. 

Our  conception  of  the  mechanism  responsible  for  the  diminution 
in  urinary  constituents  as  reported  by  Baer  and  Blum  also  furnishes 
a  reasonable  explanation  for  the  toxicity  of  tartaric  acid  observed 
by  these  investigators.  In  their  last  paper5  upon  the  subject  tar- 
taric acid  action  is  discussed  as  follows,  "Weiterhin  besass  die 
Saure  eine  erhebliche  Giftigkeit.  Ohne  bemerkenswerte  Symtome 
starb  die  Mehrzahl  unserer  Hunde  kurz  nach  Beendigung,  einzelne 
Tiere  sogar  vor  Beendigung  des  Versuchs.  Immerhin  glauben  wir 
diese  giftige  Wirkung  als  etwas  Akzidentelles  auffassen  zu  diirfen,  v 
nicht  als  die  Ursache  des  Einflusses  auf  Zucker-,  Stickstoff-  und 
Acidosekorperausscheidung. " 

SUMMARY. 

The  observation  of  Baer  and  Blum  that  sodium  tartrate  subcu- 
taneously injected  may  greatly  diminish  the  output  of  nitrogen  and 
dextrose  in  the  urine  of  phlorhizinized  dogs  has  been  substantiated 
by  the  results  of  our  investigation  on  the  subject,  but  we  differ 
from  these  authors  in  the  interpretation  of  the  phenomena  provoked. 

Our  experience  shows  that  sodium  tartrate  subcutaneously  admin- 
istered to  phlorhizinized  rabbits  and  dogs  induces  distintegrative  changes 

5  Baer  and  Blum:  Arch.  f.  exp.  Path.  u.  Pharrn.,  lxv,  p.  16,  1911. 


126     Sodium  Tartrate  and  Phlorhizin  Diabetes 

in  the  kidney  tubuli  sufficient  to  account  for  the  lessened  elimination 
of  urinary  nitrogen  and  dextrose,  observed  by  Baer  and  Blum. 

Under  strictly  comparable  experimental  conditions  similar  results 
may  be  obtained  in  animals  that  have  not  received  phlorhizin,  thus 
demonstrating  that  sodium  tartrate  acts  specifically  in  this  direc- 
tion and  that  phlorhizin  probably  contributes  little  or  nothing  to 
the  detrimental  influence  under  discussion. 


******  (TOm  T«  J(n,„„A,  M  ^  xjii  ^  i9[2 


FEEDING  EXPERIMENTS  WITH  FAT-FREE  FOOD 
MIXTURES.1 

Bv  THOMAS  B.  OSBORNE  and  LAFAYETTE  B.  MENDEL 
With  the  Cooperatzon  oe  Edna  L.  Ferby 

(Received  for  publication,  May  20,  1912.) 

Ca^ates 

ents  of  the  diet.  I  * her  them  T  V^8™1^  comP°- 
food  intake  nutrit  on  solt  Z f  '^^^ 
however  liberal  the  energy  valu  s  "  th eC°meSnotably  defective, 
be.  The  ketonuria  ano T  u  I  remammg  nutriment  may 
Phenomena  when  carbohvdt  ^  aPP6al"  a'°ng  with  other 
familiar;  and  thTneSfofSS  at'°n  ^^'^y  fails  are 
tal  postulates  of  phystlogy  °n<!  °f  *he  fundamen" 

available.   Fatea  e  of  coot  T* n° definite ^formation 

or  lesser  -m^^^S^STSft  *  « 

resent  an  indispensable  need  of  ,       at  6Xtent  the-y  reP" 

Thfi  reason  why  this  apparemtJv  fn  ^  1111  r^ma^ns  to  be  learned, 
has  not  been  answerX^ fUndament,a  ^ti™  »  nutrition 
experimental  difficu  £        en f  ? ?  attributab'*  to  the 

-  -  e«uTof~^ 

2  Cf.  Abclerhalden,  E. :  Z^scAr.  f  vhvsiol  Ch»      i  •• 

»-./•  pnysiol.  them.,  lxxvii,  p.22,  1912. 


81 

THE  JOURNAL  OF  BZOLOGtCAL  CHEMISTRY 


VOL.  XII,  NO.  1 


82 


Growth  on  Fat-free  Food 


tempts  to  maintain  animals  on  artificially  prepared  mixtures  of 
isolated  food  substances  have,  until  lately,  met  with  little  success.3 

Associated  with  this  problem  is  the  possible  significance  of  that 
hereterogeneous  group  of  substances,  resembling  the  fats  in  certain 
physical  properties,  found  with  them  in  nature,  and  currently  des- 
ignated as  "  lipoids. "  As  representatives  of  this  category  the  phos- 
phatides, and  cholesterols,  like  the  inorganic  salts,  are  found  present 
in  some  quantity  in  every  active  cell.4  This  fact  of  itself  strongly 
suggests  for  them  some  preeminent  biochemical  importance;  but 
it  by  no  means  involves  the  necessity  of  their  being  furnished  as 
such  to  the  organism.  It  is  easily  conceivable  that  the  so- 
called  lipoids  can  be  synthesized  de  novo  by  the  animal  tissues  as 
they  unquestionably  are  by  plant  cells.  The  fact  that  the  food  sup- 
ply of  growing  organisms,  viz.,  milk  or  egg  components,  furnishes 
phosphatides  and  cholesterol  preformed  speaks  only  by  indirect 
suggestion  regarding  the  absolute  need  of  these  compounds  in  the 
diet.  Finally,  the  fact  that  in  tissues,  the  " lipoids"  are  so  closely 
associated  with  the  true  fats,  i.e.,  glycerides  of  fatty  acids,  by  no 
means  proves  that  the  biological  importance  of  the  two  groups  is 
comparable  or  their  significance  as  dietary  constitutents  the  same. 

The  question  of  the  role  of  fats  as  indispensable  factors  in  the  diet 
has  been  approached  by  Stepp.5  In  attempting  to  ascertain 
whether  animals  are  dependent  upon  their  food  supply  for  lipoids 
or  can  furnish  them  by  synthesis  like  plants,  he  fed  materials  ex- 
tracted with  ether  and  alcohol  to  mice  and  observed  the  effect  on  the 
nutritive  equilibrium  of  the  animals.  Obviously  this  method  of 
preparing  the  food  eliminated  true  fats  from  the  diet  at  the  same 
time.  Stepp's  observations  and  conclusions  deserve  to  be  care- 
fully examined  in  connection  with  the  problem  at  hand.  He  noted 
that  without  exception  his  mice  succumbed  in  a  few  weeks  when 
offered  otherwise  adequate  food  mixtures  that  had  been  thoroughly 
extracted.    The  deduction  is  made  that  the  nutritive  failure  is  due 

3  A  discussion  of  earlier  attempts  in  this  direction  will  be  found  in  our 
monograph:  Feeding  Experiments  with  Isolated  Food-Substances,  Carnegie 
Institution  of  Washington,  Publication  156,  Parts  I  and  II,  1911. 

4  For  a  general  description  of  the  so-called  lipoids,  their  occurrence  and 
possible  biochemical  significance,  see  Bang :  Ergeb.  d.  Physiol. vi,  p.  131, 1907 ; 
viii,p.  463,  1909. 

5  Stepp:  Biochem.  Zeilschr.,  xxii,  p.  452,  1909;  Verhandl.  Kongrasses  f.  inn. 
Med.,  xxviii,  p.  324,  1911 ;  Zeitschr.  f.  Biol,  lvii,  p.  135,  1911. 


Thomas  B.  Osborne  and  Lafayette  B.  Mendel  83 


to  the  lack  of  certain  " lipoid"  substances,  because  the  addition  of 
alcohol-ether  extracts  of  materials  known  to  be  rich  in  this  type  of 
compound  sufficed  to  keep  the  animals  alive.  The  lacking  sub- 
stance is  assumed  not  to  be  inorganic,  since  the  addition  of  the  ash 
of  the  lipoid  extracts  made  from  the  food  material  failed  to  main- 
tain the  mice.  Furthermore — and  this  calls  for  emphasis  here — 
the  sustaining  component  is  asserted  not  to  be  ordinary  fat  inas- 
much as  the  addition  of  so  typical  a  fat  as  butter  failed  to  replace 
the  missing  life-sustaining  factor.  The  latter  was  found  to  be  ex- 
tractable  from  skimmed  milk  rather  than  from  the  cream  fraction. 
Quoting  Stepp : 

The  life-sustaining  alcohol-ether-soluble  food  components,  in  the  absence 
of  which  mice  regularly  succumb,  are  not  fats.  This  is  shown,  aside  from  the 
experiments  in  which  butter  was  fed,  by  the  following  experiment :  On  a  diet 
of  extracted  foods  to  which  tripalmitin,  tristearin  and  triolein  are  added,  all 
the  animals  die  precisely  as  on  the  extracted  food  alone.  That  lecithin 
(Merck)  and  cholesterol  do  not  represent  the  sole  lipoids  essential  to  life  is 
shown  by  the  experiment  of  adding  them  to  the  extracted  food:  all  of  the 
animals  died . 6 

It  is  rather  difficult  to  believe  that  skimmed  milk,  at  best  very 
deficient  in  ether-alcohol  soluble  components,  should  contain  an 
eminently  important  lipoid  in  any  adequate  amount  while  other 
materials,  like  butter,  which  must  contain  some  compounds  of  this 
type  are  inadequate.  However,  certain  of  these  lipoids  are  doubt- 
less highly  sensitive  to  chemical  change;  so  that  it  is  conceivable 
that  they  lose  their  physiological  potency  through  chemical  manip- 
ulation. Furthermore  the  recent  experiences  with  beri-beri  and 
other  forms  of  peripheral  neuritis  have  emphasized  how  small 
may  be  the  actual  amount  of  a  specific  substance  which  determines 
proper  physiological  functioning.7 

Stepp's  experiments  on  mice  by  no  means  solve  the  question 
which  we  have  raised  at  the  outset  with  regard  to  the  necessity  of 
fats  in  the  diet.  They  furnish  no  evidence  that  the  "lipoid'  'mix- 
tures which  he  employed  to  maintain  or  resuscitate  his  animals 
were  actually  free  from  true  fats,  though  the  quantities  in  some  cases 
(such  as  the  experiments  with  extract  of  skimmed  milk)  must  at 

6  Stepp:  Zeitschr.  f.  Biol,  lvii,  p.  170,  1911. 

7  Cf.  Funk:  Journ.  of  Physiol, xliii,  p.  395,  1911. 


84 


Growth  on  Fat-free  Food 


best  have  been  exceedingly  small.  Other  occasional  experiments 
in  the  literature8  are  of  too  brief  duration  to  settle  the  point. 

Employing  the  methods  which  were  adopted  in  our  earlier  feeding 
experiments  with  isolated  food  substances9  we  have  succeeded  in 
inducing  a  normal  rate  of  growth  in  white  rats  with  dietaries  devoid 
of  fat  throughout  almost  the  entire  period  during  which  growth 
ordinarily  continues.10  The  foods  were  prepared  by  mixing  carefully 
isolated  and  purified  proteins  with  starch,  sugar  and  "protein-free 
milk,"11  the  latter  having  first  been  thoroughly  extracted  with 
ether.  The  starch  was  stirred  with  water,  heated  until  the  grains 
were  ruptured  and  then  the  other  ingredients  thoroughly  mixed 
with  the  starch  paste,  and  afterwards  dried  in  a  current  of  hot  air 
until  thin  cakes  of  desiccated  food  were  obtained.  These  were  then 
fed,  along  with  the  water,  to  the  rats  kept  in  the  metabolism  cages 
devised  for  these  studies.12  Although  the  foods  may  certainly  be 
designated  as  fat-free,  it  is  perhaps  not  permissible  to  speak  of  them 
as  lipoid-free;  for  according  to  the  current  definition,  the  so-called 
" lipoids"  include  substances  soluble  in  hot  alcohol  which  may  not 
dissolve  in  ether.  None  of  our  isolated  food  materials  were  sub- 
jected to  extraction  with  hot  alcohol.  Undoubtedly  such  treat- 
ment would  remove  other  substances  as  well  as  lipoids  from  such 
a  mixture  as  the  " protein-free  milk."  This  fact  deserves  to  be 
emphasized,  as  does  the  necessity  of  conducting  ether  extractions 
under  appropriate  conditions.  When,  for  example,  a  specimen  of 
air-dry  " protein-free  milk"  was  extracted  with  ordinary  ether  it 

8  Cf.  Lummert :  Pfliiger's  Archiv,  lxxi,  p.  176,  1898. 

9  Cf.  Osborne,  T.  B.,  and  L.B.Mendel:  Feeding  Experiments  with  Isolated 
Food-Substances,  Carnegie  Institution  of  Washington,  Publication  156,  Parts 
I  and  II,  1911. 

10  In  these,  as  in  all  our  other  experiments  in  which  very  young  animals 
exhibited  a  normal  rate  of  growth  on  mixtures  of  isolated  food-substances, 
we  have  not  yet  succeeded  in  bringing  the  animals  to  their  maximum  normal 
size  on  the  dietaries  employed.  This  failure  to  attain  complete  growth  in- 
volves some  factor  in  nutrition  other  than  the  fat  and  is  at  present  under 
investigation. 

11  For  the  character  of  this  product  cf.  Osborne,  T.  B.,  and  L.  B.  Mendel: 
Feeding  Experiments  with  Isolated  Food-Substances,  Carnegie  Institution  of 
Washington,  Publication  156,  Parts  I  and  II,  1911;  and  Science,  xxxiv,  p.  722, 
1911. 

12  Cf.  Osborne,  T.  B.,  and  L.  B.  Mendel:  Zeitschr.  f.  biolog.  Technik  and 
Methodik,  1912. 


86 


Growth  on  Fat-free  Food 


yielded  over  2  per  cent  of  extract;  but  the  same  product  carefully 
dried  in  hydrogen  and  extracted  with  anhydrous  ether  yielded  only 
0.13  per  cent  of  ether  extract,  which  was  not  increased  when  an 
alkali  solution  of  the  substance  was  shaken  out  with  benzine  and 
ether  according  to  the  method  commonly  applied  to  milk  pow- 
ders. Actual  extraction  of  the  foods  used  by  us  yielded  not  more 
than  an  insignificant  trace  of  ether  extract. 


Chart  3.  Rat  533,  9  ;  Chart  4,  Rat  661,  d*.  The  food  during  the  casein- 
fat-free  period  had  the  following  percentage  composition : 


 ;   22.0  .  i 

Sucrose   20.0  .  mjj 

Starch  '.   28.5 

"Artificial"  protein-free  milk"   29. 5 

100.0 

Illustrative  charts  of  our  feeding  trials  are  introduced  here.  The 
ordinates  of  the  curves  represent  body-weight  (solid  line)  or  food 


13  The  successful  use  of  this  purely  artificial  product  consisting  of  Ca,  1.97; 
Mg,  0.23 ;  Na,  2.03 ;  K,  2.66 ;  P04, 3.33 ;  CI,  4. 13 ;  S04, 0.30 ;  Fe,  0.04 ;  Citric  acid, 
3.33;  Lactose,  82.0  per  cent,  has  been  described  by  Osborne  and  Mendel; 
Proc.  Soc.  of  Exp.  Biol,  and  Med.,  ix,  p.  73,  1912.  The  relatively  early  fail- 
ure to  continue  to  grow,  shown  by  chart  3,  was  caused  by  diseased  lungs 
which  terminated  the  life  of  Rat  533. 


Thomas  B.  Osborne  and  Lafayette  B.  Mendel  87 


intake  (dotted  line)  in  grams;  the  abscissae  represent  days.  The 
average  (normal)  curve  of  growth,  plotted  from  body-weight  data 
available  for  normally  growing  animals  of  the  same  sex,  is  repre- 
sented by  a  broken  line  for  comparison.  In  period  1  of  all  curves 
the  rats  were  fed  on  ordinary  mixed  diet  or  by  the  mother. 


?1 

0 

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Days 

Chart  5,  Rat  529,  cf .  The  fat-free  diet  had  the  following  percentage  com- 
position: 


Casein  

Edestin  

Sucrose  

Starch  

"Artificial"  protein-free  milk 


Period  2 
22.0 
0.0 
.  20.0 
.  28.5 
.  29.5 

100.0 


Period  S 
0.0 
22.0 
20.0  . 
28.5 
29.5 

100.0 


In  so  far  as  one  can  judge  by  appearance  and  body  weight  these 
experiments  with  fat-free  diets  show  growth  quite  as  successful  as 


88 


Growth  on  Fat-free  Food 


that  attained  with  natural  or  artificial  mixtures  of  all  the  types  of 
food  stuffs.  Although  we  cannot  claim  a  complete  freedom  from 
" lipoids"  for  the  foods  prepared  as  described  above,  it  is  scarcely 

likely  that  products  so  carefully 
isolated  can  include  any  signifi- 
cant quantities  of  cerebrosides  or 
phosphatides.  This  is  peculiarly 
true  of  experiment  6  in  which  the 
sole  possibilities  of  contamination 
are  associated  with  the  recrystal- 
lized  phosphorus-free  protein 
edestin  and  refined  starch. 

McCollum14  has  demonstrated 
that  the  phosphorus  needed  by  an 
animal  for  phosphatide  forma- 
tion can  be  drawn  from  inorganic 
phosphates,  and  that  phospha- 
tides can  be  synthesized  anew  in 
the  animal  body.  Rohmann 15 
asserts  the  possibility  of  lecithin 
synthesis  in  mice  which  were 
maintained  into  the  second  gen- 
eration on  lecithin-free  food.  Our 
own  experiments  point  in  the 
same  direction  with  regard  to 
the  lipoids  in  general;  and  they 
give  positive  evidence  of  the  dis- 
pensableness  of  true  fats  for 
growth.16 

14  McCollum:  Amer.  Journ.  of  Physiol, xxv,  p.  120,  1909;  McCollum  and 
Halpin:  This  Journal,  xi,  1912,  Proc.  Soc.  Biol.  Chem.,  p.  xiii;  also  Fingerling: 
Biochem.  Zeitschr.,  xxxviii,  p.  438, 1912. 

15  Rohmann :  Biochemie,  1908,  p.  109. 

16  In  agreement  with  Stepp,  we  have  not  yet  succeeded  similarly  in  induc- 
ing adequate  growth  in  mice  with  similar  diets.  Stepp,  who  used  crude  food 
substances,  is,  however,  cautious  in  his  statements.  He  says :  "  Wenn  nach 
den  mitgeteiltenVersuchen  und  den  anschliessenden  Erorterungen  der  Schluss 
sich  aufdrangt,  dass  gewisse  alkohol-atherldsliche  Substanzen  fur  die  Erndh- 
rung  von  Mausen '  unentbehrlich  sind,  so  mochte  ich  diesen  Schluss  nicht  ohne 
eine  Einschninkung  aufrechterhalten.    Die  Untersucher,  die  sich  mit  dem 


Chart  6,  Rat  640,  & .  The  fat- 
free  food  had  the  following  per- 
centage composition: 

Edestin   22.0 

Sucrose   20.0 

Starch   28.5 

"Artificial"  protein  free-milk  .  29.5 


100.0 


Thomas  B.  Osborne  and  Lafayette  B.  Mendel  89 

The  possibilities  of  the  method  of  study  introduced  by  us  are 
manifest.  The  problems  of  the  origin  of  fats  in  animals  and  their 
genesis  from  various  carbohydrates  or  proteins  are  thus  made  ap- 
proachable by  experiment.17  We  hope  to  return  to  these  ques- 
tions later. 


Studium  der  Lipoide beschaf tigten,  haben,  wie  schon  kurz  erwahnt,  an  diesen 
Korpen  Eigenschaften  gefunden,  die  man  in  der  Chemie  bisher  kaum 
kannte.  Die  Lipoide  haben  eine  ganz  ausserordentliche  Fahigkeit,  auf  die 
Loslichkeit  anderer  Stoffe  einzuwirken  und  ihnen  Loslichkeit  in  den  spezi- 
fischen  Lipoidlosungsmitteln  zu  verleihen,  in  denen  die  Stoffe  sonst  ganzlich 
unloslich  sind.  So  ware  es  nicht  undenkbar,  dass  gemeinschaftlich  mit  den 
Lipoiden  irgendwelche  unbekannte  lebenswichtige  Stoffe  in  Losung  gehen 
und  dass  so  die  Lipoide  gewissermassen  zu  Tragern  f  ur  diese  Stoffe  wi'irden, 
dass  mit  anderen  Worten  bei  der  Entfernung  von  Lipoiden  die  unbekannten 
Korper  mit  entfernt  und  bei  Zusatz  von  Lipoiden  mit  diesen  zugesetzt  wer- 
den.  Ein  Hinweis  auf  eine  derartige  Moglichkeit  erscheint  notwendig,  so- 
lange  es  nicht  gelingt,  die  Versuche  mit  chemisch  reinen  Korpern  durchzu- 
fiihren." 

l7Lummert  (Pfliiger's  Archiv,  lxxi,  p.  176,1898)  has  made  attempts  in  the 
same  direction. 


Reprinted  from  Tun  Journai,  or  Biological  Chemistry  Vol.  XII,  No.  3,  1912 


THE  ROLE  OF  GLIADIN  IN  NUTRITION.1 

By  THOMAS  B.  OSBORNE  and  LAFAYETTE  B.  MENDEL, 

With  the  Cooperation  of  Edna  L.  Ferry. 

(From  the  Laboratories  of  the  Connecticut  Agricultural  Experiment  Station 
and  the  Sheffield  Laboratory  of  Yale  University,  New  Haven,  Connecticut.) 

(Received  for  publication,  July  31,  1912.) 

Our  notions  regarding  the  relation  of  the  food  proteins  to  tissue 
proteins,  and  the  role  of  proteins  in  nutrition  have  experienced 
radical  changes  in  recent  years.  Side  by  side  with  the  increasing 
evidence  of  distinct  structural  differences  between  the  albuminous 
compounds  of  different  origin  and  the  chemical  dissimilarity  which 
may  even  characterize  two  proteins  derived  from  a  common  source, 
such  as  some  particular  seed,  has  arisen  the  well  founded  conviction 
that  it  is  impossible  to  develop  marked  changes  in  the  character 
of  the  tissues  of  animals  correlated  with  the  character  of  the  food 
ingested.  Whatever  may  be  the  source,  or  chemical  make-up,  of 
the  latter  previous  to  its  involvement  in  the  nutritive  processes, 
the  resulting  tissue  cells  and  fluids  remain  characteristic  and 
specific  for  the  species.  "Der  Artcharakter  wird  durch  die  Art 
der  Ernahrung  nicht  beeinnusst"  (Abderhalden). 

How  this  possibility  of  the  fixity  of  the  tissues  in  the  midst  of 
diversity  of  food  types  results  is  made  apparent  by  the  newer 
knowledge  respecting  the  role  of  digestion  in  nutrition.  The 
structural  pesuliarities  which  determine  the  individuality  of  the 
proteins  are  lost  by  the  digestive  process;  hence  we  have  ultimately 
to  deal  with  the  fragments  of  the  original  complexes  in  the  problems 
pertaining  to  nutrition.  Our  food  stuffs  are  currently  assumed  to 
leave  the  alimentary  tract  largely,  if  not  entirely,  in  the  form  of 

1  The  expenses  of  this  investigation  were  shared  by  the  Connecticut 
Agricultural  Experiment  Station  and  the  Carnegie  Institution  of  Washing- 
ton. 

473 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  XII,  NO.  3. 


474 


Gliadin  in  Nutrition 


the  so-called  ammo-acid  "Bausteine."  It  is  these  which  become 
our  immediate  concern  in  the  intermediary  problems  of  metabo- 
lism that  result  in  the  construction  or  renewal  of  the  specific  body 
protein.  Quoting  Abderhalden:  "Unsere  Korperzellen  erfahren 
niemals,  welcher  Art  die  aufgenommene  Nahrung  war." 

In  the  organism  proper  the  proteins,  as  such,  may  be  responsible 
for  various  physiological  functions.  "At  present  we  cannot  fully 
comprehend  the  role  of  the  proteins,  but  we  must  assume  that 
many  of  the  enigmatical  properties  of  living  matter  depend  on 
this  activity  of  intact  protein  molecules.  We  can  obtain  some 
idea  of  the  possible  variety  in  the  combinations  of  the  protein 
Bausteine  by  recalling  the  fact  that  they  are  as  numerous  as  the 
letters  in  the  alphabet  which  are  capable  of  expressing  an  infinite 
number  of  thoughts.  Every  peculiarity  of  species  and  every 
occurrence  affecting  the  individual  may  be  indicated  by  special 
combinations  of  protein  Bausteine,  that  is  to  say  by  specific  pro- 
teins. Consequently  we  may  readily  understand  how  peculiarity 
of  species  may  find  expression  in  the  chemical  nature  of  the  pro- 
teins constituting  living  matter,  and  how  they  may  be  transmitted 
through  the  material  contained  in  the  generative  cells."2  As  one 
of  us  has  written  earlier:  "The  results  of  my  work  have  shown 
that  no  two  seeds  are  alike  in  their  protein  constituents,  and  that 
those  proteins  which  appear  to  be  alike  are  found  only  in  seeds 
that  are  botanically  closely  related.  As  I  have  elsewhere  pointed 
out,  it  would  seem  that  these  differences  in  the  reserve  food  sub- 
stances of  the  endosperm  must  have  an  important  bearing  on  the 
character  of  the  developing  embryo  which  derives  its  first  food 
from  them.  This  food  substance,  and  the  embryo  as  well,  are 
the  final  products  of  the  series  of  chemical  changes  which  led  to 
their  formation.  When  the  embryo  begins  its  development  it 
finds  at  hand  a  definite  food,  which  for  each  individual  of  the  same 
species  is  the  same,  but  for  the  individuals  of  different  species  is 
different.  Each  member  of  a  species  begins  its  independent  life 
under  similar  chemical  conditions,  but  under  chemical  conditions 
which  are  different  from  those  of  every  other  species.  When, 
therefore,  each  individual  plant  reaches  that  stage  of  development 
at  which  its  organs  of  assimilation  are  able  to  furnish  it  with  nutri- 

2Kossel:  Lectures  on  the  Herter  Foundation.  The  Proteins.  Johns 
Hopkins  Hospital  Bulletin,  xxiii,  p.  76,  1912. 


T.  B.  Osborne  and  L.  B.  Mendel  475 


ment  from  its  external  surroundings,  it  is  highly  probable  that 
its  chemical  processes  have  already  been  established  along  definite 
lines  which  it  must  follow  throughout  the  rest  of  its  life."3 

In  the  preliminary  processes  of  metabolism,  however,  the  char- 
acter of  the  amino-acid  fragments  apparently  assumes  a  dominating- 
importance.  The  modern  chemistry  of  the  proteins  has  dis- 
closed the  fact  that  the  variations  between  the  different  albumi- 
nous compounds  in  respect  to  their  Bausteine  may  be  both  quanti- 
tative and  qualitative  in  character.  This  has  raised  the  question 
of  the  relative  physiological  value  of  unlike  proteins.  "The  fact 
that  so  many  of  the  vegetable  proteins,  which  serve  extensively 
as  food,  have  been  shown,  by  our  present  investigation,  to  yield 
such  different  proportions  of  the  various  nitrogenous  decomposi- 
tion products,  as  compared  with  the  animal  proteins,  makes  it 
a  matter  of  the  greatest  interest  and  importance  to  know  some- 
thing more  of  the  processes  involved  in  this  synthesis."4 

Whether  protein  can  be  suitably  utilized  when  administered 
in  its  completely  digested  or  abiuret  form  as  well  as  in  its  natural 
condition  need  not  concern  us  here;  since  the  possibility  of  main- 
taining individuals  in  satisfactory  nutritive  balance,  at  least  for 
a  not  inconsiderable  time,  on  an  intake  made  up  exclusively  of 
Bausteine  has  been  demonstrated.  It  would  seem,  therefore,  as 
if  the  problem  of  replacing  the  larger  protein  complexes  by  their 
elementary  constituent  fragments  had  been  to  a  certain  extent 
solved.5  If  we  assume,  in  harmony  with  some  of  the  prevailing 
views  of  metabolism,  and  notably  that  supported  by  Abderhalden, 
that  the  animal  must  construct  its  tissue  proteins  from  the  amino- 
acid  fragments  which  are  furnished  by  protein  hydrolysis,  it  is 
obvious  that  deficiencies  in  quantity  in  the  Bausteine  or  a  lack 
of  one  or  more  of  them  must  lead  to  serious  nutritive  disturbances. 
The  chemical  fixity  of  the  tissues  under  widely  differing  nutrient 
environment  points  in  the  same  direction.  Abderhalden  has 
maintained  that,  so  long  as  there  is  no  evidence  that  amino-acids 
can  readily  experience  a  transformation  into  one  another  in  the 
organism,  the  extent  of  protein  construction  in  the  body  must  be 

3  Osborne:  Proc.  Soc.  Exp.  Biol,  and  Med.,  v,  p.  105,  1908. 
'Osborne  and  Harris:    Jour.  Amer.  Chem.  Soc,  xxv,  p.  323,  1903. 
5  Cf .  Abderhalden :  Synthese  der  Zellbausteine  in  Pflanze  und  Tier, 
Berlin,  1912. 


476 


Gliadin  in  Nutrition 


limited  by  the  amino-acid  which  is  present  in  the  smallest  relative 
amount  in  our  intake.  The  fact  that  certain  proteins,  such  as 
gelatin  and  zein,  which  are  notably  defective  in  respect  to  the 
number  of  the  amino-acids  which  they  yield,  are  unable  by  them- 
selves to  promote  nutritive  equilibrium  and  supply  the  nitrogenous 
needs  of  the  diet  might  be  quoted  in  support  of  the  views  mentioned 
above.  If  Abderhalden's  hypothesis  regarding  the  nature  of  protein 
metabolism  is  correct  it  follows  that  those  food  proteins  which 
approach  most  nearly  to  the  tissue  proteins  in  their  amino-acid 
make-up  should  most  easily  supply  the  protein  needs  of  the  animal. 
Michaud6  has  undertaken  to  demonstrate,  in  accord  with  this, 
that  the  protein  minimum  of  dogs  can  be  maintained  at  a  lower 
level  when  the  intake  is  in  the  form  of  dog  tissue  than  in  the  form 
of  proteins  differing  widely  therefrom  in  their  chemical  make-up; 
yet  the  investigations  heretofore  recorded  with  these  proteins  lead 
to  the  belief  that  they  are,  at  least  to  some  degree,  utilized  as  food 
by  the  animal;  even  when  they  are  fed  as  the  sole  source  of  nitrogen. 
Some  of  these  proteins  lacking  one  or  more  of  the  cleavage  prod- 
ucts known  to  be  necessary  for  the  formation  of  the  proteins  of 
the  animal  body  are  of  relatively  high  efficiency  in  preventing 
loss  of  body  nitrogen  due  to  endogenous  metabolism,  although 
they  are  insufficient  for  growth.7  It  is  evident  that  "the  processes 
of  replacing  nitrogen  degraded  in  cellular  metabolism  are  not  of 
the  same  character  as  the  processes  of  growth.  It  seems  also 
to  be  a  necessary  conclusion  that  the  processes  of  cellular  cata- 
bolism  and  repair  do  not  represent  a  series  of  chemical  changes 
involving  the  destruction  and  reconstruction  of  an  entire  protein 
molecule."8  Regarding  the  necessity  of  distinguishing  carefully 
between  maintenance,  repair  and  growth  in  nutrition  we  shall 
have  more  to  say  later.  Undoubtedly  the  failure  to  bear  these 
distinctions  in  mind  has  led  to  much  confusion  in  the  past.  Fur- 
thermore, investigators  have  heretofore  been  so  largely  concerned 
with  the  functions  of  proteins  as  a  whole  in  important  biological 
processes  that  the  possibility  of  their  individual  participation 

6  Michaud:  Zeitschr.  f.  physiol.  Chem.,  lix,  p.  405,  1909;  cf.  also  Frank 
and  Schittenhelm :  ibid.,  lxx,  p.  99,  1910;  lxxiii,  p.  157,  1911. 

7  Cf .  Osborne  and  Mendel :  Carnegie  Institution  of  Washington,  Publi- 
cation 156,  pt.  ii,  1911;  also  Zeitschr.  f.  physiol.  Chem.,  1912  (in  press). 

8McCollum:  Amer.  Journ.  of  Physiol.,  xxix,  p.  215,  1911. 


T.  B.  Osborne  and  L.  B.  Mendel  477 


and  use  has  been  generally  overlooked.  As  Kossel  lias  lately 
said:  "Hitherto  the  appearance  of  protein  Bausteine  in  the  living 
organism  has  always  been  ascribed  to  protein  decomposition. 
But  this  supposition  is  unjustified.  We  must  rather  assume  that 
these  Bausteine  may  appear  and  disappear  in  the  body  without 
at  any  time  forming  part  of  a  protein  molecule.  And  further  we 
may  suppose  that  only  under  certain  circumstances,  for  definite 
physiological  purposes,  are  these  independent  groups  stored  in  a 
collected  form — the  protein  substances."9 

Attempts  have  been  made  at  various  times  in  the  past  to  perfect 
the  so-called  abnormal  or  incomplete  proteins  by  adding  to  them 
in  the  diet  one  or  more  ammo-acids  which  are  known  to  be  lacking 
from  the  complex.  This  is  true  of  studies  made  with  gelatin— 
which  yields  no  tyrosine,  tryptophane  or  cystine — and  with  zein, — 
a  protein  which  yields  no  tryptophane,  and  from  which  no  lysine 
or  glycocoll  can  be  obtained.  These  trials  have,  all  in  all,  not 
been  very  satisfactory.  Other  experiments  in  which  an  amino- 
acid,  such  as  tryptophane,  has  been  intentionally  eliminated  from 
the  food  mixture  have  speedily  exhibited  a  nutritive  defect  in 
the  dietary.  In  any  event  it  seems  clear,  from  such  evidence  as 
is  available  at  the  present  time,  that  the  cyclic  compounds,  tyro- 
sine, phenylalanine,  histidine  and  tryptophane,  are  indispensable 
for  the  welfare  of  the  organism.  Indeed  W.  A.  Osborne  has 
expressed  the  view  that  the  essential  difference  between  the  animal 
and  the  plant  organism  lies  in  their  respective  ability  or  inability 
to  synthesize  substances  of  the  cyclic  type.  Cyclopoiesis,  accord- 
ing to  him,  is  a  property  exhibited  solely  by  the  vegetable 
organism. 

Are  the  other  amino-acids  equally  indispensable?  At  the 
present  moment  it  is  impossible  to  give  any  definite  answer  to 
the  question  as  to  whether  an  amino-acid  like  leucine,  for  example, 
can  be  replaced  by  alanine,  or  any  other  closely  related  form.  In 
one  case,  in  any  event,  the  possibility  of  a  synthesis  of  an  amino- 
acid  de  novo  in  the  animal  organism  has  been  admitted.  Prolonged 
feeding  experiments  with  casein  from  which  glycocoll  has  not 
been  obtained,  as  well  as  the  enormous  production  of  glycocoll 
for  the  hippuric  acid  synthesis  after  the  administration  of  benzoic 

•Kossel:  Lectures  on  the  Herter  Foundation.  The  Proteins.  Johns 
Hopkins  Hospital  Bulletin,  xxiii,  p.  76,  1912. 


478 


Gliadin  in  Nutrition 


acid10 — a  production  out  of  all  proportion  to  the  assumed  content 
of  preformed  glycocoll  in  the  food  intake,  or  the  body  tissues 
themselves — leave  little  doubt  of  the  capacity  of  the  animal 
cell  to  synthesize  at  least  one  amino-acid. 

For  evidence  of  the  formation  of  amino-acids  more  complex 
than  glycocoll,  the  recorded  experiments  with  gliadin  must  be 
taken  into  consideration.  This  substance,  an  alcohol-soluble  pro- 
tein of  the  prolamine  type,  possesses  a  special  interest  in  that  it 
yields  so  little  of  the  diamino-acid,  lysine,  as  well  as  of  glycocoll, 
that  these  have  not  heretofore  been  obtained  from  it  by  the  usual 
analytical  methods.  It  furthermore  contains  a  relatively  small 
proportion  of  both  arginine  and  histidine,  and  extremely  large 
proportions  of  glutaminic  acid  and  ammonia  yielding  groups. 
Its  ready  digestibility  has  been  demonstrated  repeatedly11  in 
contrast  to  the  greater  resistance  of  the  "abnormal"  protein  zein. 
Henriques12  reported  that  he  kept  rats  in  nitrogenous  equilibrium 
on  a  diet  in  which  gliadin  constituted  the  sole  form  of  nitrogenous 
intake,  although  he  failed  when  the  tryptophane-free  zein  was  used. 
Abderhalden  and  Funk13  were  similarly  successful  with  gliadin 
fed  to  dogs.  They  state,  however,  that  in  one  case  the  preparation 
of  gliadin  fed  by  them  contained  0.35  per  cent  of  lysine;  and  they 
intimate  that  the  nutritive  equilibrium  secured  by  Henriques  on 
a  diet  containing  gliadin  as  the  sole  protein  was  due  to  an  impure 
preparation.  The  question  of  lysine  synthesis  in  the  body  is 
expressed  by  Rona14  as  follows:  "Das  Problem  is  also  durch 
die  Versuche  von  Henriques  noch  nicht  gelost,  hingegen  sprechen 
alle  unsere  Erfahrungen  dafur  dass  die  Aminosauren,  Glykokoll 
ausgenommen,  im  Organismus  nicht  neugebildet  werden."  In 
other  experiments  Henriques  and  Hansen15  have  stated  that 
they  were  able  to  get  rats  into  a  state  of  nitrogenous  equilibrium 
on  a  diet  containing  the  nitrogen  solely  in  the  form  of  the  mono- 
amir.o  fraction  of  a  digest.    Here  too,  as  in  the  experiments  with 

10  Cf.  Magnus-Levy:  Biochem.  Zeitschr.,  vi,  p.  523,  1907;  Ringer:  this 
Journal,  x,  p.  327,  1911;  Epstein  and  Bookman:  ibid.,  x,  p.  353,  1911. 

11  Cf.  Mendel  and  Fine:  this  Journal,  x,  p.  303,  1911. 

12  Henriques:  Zeitschr.  f.  physiol.  Chem.,  lx,  p.  105,  1909. 

13  Abderhalden  and  Funk:  ibid.,  lx,  p.  418,  1909. 

14  Rona:  Oppenheimer's  Handbuch  der  Biochemie,  iv,  pt.  i,  p.  550. 

15  Henriques  and  Hansen:  Zeitschr.  f.  physiol.  Chem.,  xliii,  p.  417,  1904; 
and  xlix,  p.  113.  1906. 


T.  B.  Osborne  and  L.  B.  Mendel  479 


gliadin,  a  synthesis  of  nitrogenous  compounds  of  the  type4,  precipi- 
table  by  phosphotungstic  acid  must  be  assumed  if  the  nutritive 
equilibrium  of  the  experimental  animals  was  at  all  adequate.  It 
will  be  seen,  therefore,  that  the  problem  of  di-amino  synthesis 
has  heretofore  largely  hinged  upon  the  validity  of  the  work  of 
Henriques. 

The  situation  has  been  summed  up  by  Rona  in  these  words: 
"Vorlaufig  mussen  wir  also  daran  festhalten,  daes  eine  Ueber- 
fuhrung  einer  Aminosaure  in  eine  andere,  bezw.  eine  Neubildung 
einer  Aminosaure  (Glykokoll  ausgenommen)  im  tierischen  Organ- 
ismus  nicht  stattfindet."16 

EXPERIMENTAL  PART. 

Employing  the  methods  which '  we  have  developed  in  recent 
years  in  connection  with  our  feeding  experiments  with  isolated 
food  substances17  we  have  accumulated  a  large  number  of  data 
which  refer  directly  to  the  nutrient  r61e  of  gliadin  in  the  animal 
organism.  Inasmuch  as  we  have  succeeded,  by  the  application 
of  care  in  the  management  of  the  rats,  by  furnishing  suitable 
hygienic  environment  and  appropriately  selected  diet,  in  main- 
taining these  animals  in  good  nutritive  condition  on  mixtures  of 
isolated  food  stuffs  over  periods  of  more  than  500  days,  we  believe 
that  some  of  the  criticisms  which  have  been  aimed  at  experiments 
carried  out  on  rats  are  thereby  met.  The  guiding  considerations 
which  have  led  to  the  special  proportions  of  nutrients,  etc.,  in  the 
food  mixtures  reported  below  have  been  discussed  in  some  detail 
in  our  previous  publications.18  The  upshot  of  our  trials  has  been 
the  demonstration  that  the  gliadins  of  wheat  and  rye,  as  well  as 
the  closely  related  alcohol-soluble  hordein  of  barley — all  of  which 
are  similar  in  the  proportion  of  their  Bausteine — suffice  for  the 
maintenance  of  rats  without  growth. 

16  Rona:  Oppenheimer's  Handbuch  der  Biochemie,  iv,  pt.  i,  p.  550. 

17  Osborne  and  Mendel:  Carnegie  Institution  of  Washington,  Publi- 
cation 156,  pts.  i  and  ii,  1911;  Zeitschr.  f.  biol.  Technik  u.  Methodik,  ii,  p. 
313,  1912;  and  Zeitschr.  f.  physiol.  Chem.,  1912  (in  press). 

18  Osborne  and  Mendel:  Carnegie  Institution  of  Washington,  Publi- 
cation 156,  pt.  ii,  1911;  Science,  N.  S.,  xxxiv,  p.  722,  1911;  Zeitschr.  f.  physiol. 
Chem.,  1912  (in  press). 


Gliadin  in  Nutrition 


Preparation  and  composition  of  gliadin. 

The  gliadin  used  for  these  feeding  experiments  was  made  from 
very  thoroughly  washed  wheat  gluten  from  which  all  proteoses 
and  other  water-soluble  proteins  had  been  removed  as  completely 
as  possible.  The  alcoholic  extract  of  this  gluten  was  filtered  water- 
clear,  thereby  separating  any  suspended  glutenin  or  other  proteins 
insoluble  in  70  per  cent  alcohol.  After  concentrating  the  alcoholic 
extract  the  residual  gliadin  was  dissolved  in  alcohol,  and  its  solution 
poured  in  a  thin  stream  into  a  very  large  volume  of  cold  water, 
thereby  removing  any  water-soluble  substance  which  might  possi- 
bly be  set  free  when  the  gluten  was  dissolved.  The  precipitated 
gliadin  was  again  dissolved  in  alcohol,  and  its  syrupy  solution  poured 
into  a  very  large  quantity  of  absolute  alcohol,  and  thus  precipitated 
as  a  coherent  mass.  This  was  then  digested  with  fresh  quanti- 
ties of  absolute  alcohol,  and  finally  with  ether  and  was  easily 
reduced  to  a  powder.  After  drying  in  the  air,  the  gliadin  thus  ob- 
tained formed  a  snow  white  powder  which  was  completely  soluble 
in  70  per  cent  alcohol.  It  is  difficult  to  see  how  gliadin,  thus 
prepared,  can  contain  any  other  proteins  than  those  soluble  in 
alcohol,  or  how  any  purer  preparation  could  be  made. 

As  it  was  of  the  greatest  importance  to  know  whether  or  not 
this  gliadin  was  entirely  free  from  lysine,  we  made  a  very  careful 
examination  of  two  portions  of  100  grams  each,  according  to  the 
method  of  Kossel  and  Kutscher,  with  which  we  have  had  extensive 
experience.  The  result  was  in  each  case  entirely  negative,  corre- 
sponding with  our  earlier  experience,  as  well  as  with  that  of  Kossel 
and  Kutscher  and  of  Abderhalden.  However,  in  view  of  the  rela- 
ti  ely  considerable  precipitate  produced  by  phosphotungstic  acid,  it 
seemed  possible  that  some  lysine  might  be  contained  therein,  under 
conditions  which  rendered  its  separation  as  the  picrate  difficult. 
This  seemed  the  more  probable  in  view  of  our  previous  experience 
in  attempting  to  isolate  lysine  as  the  picrate  directly  from  the 
products  of  the  hydrolysis  of  casein.  In  this  attempt  we  obtained 
less  than  one-half  as  much  as  by  Kossel  and  Kutscher's  method, 
thus  showing  the  effect  of  the  presence  of  other  amino-acids. 

We  accordingly  made  renewed  efforts  to  obtain  lysine  picrate 
from  our  solutions*.  In  one  case  fractional  precipitation  with 
phosphotungstic  acid  was  employed  without  success.    In  the  other 


T.  B.  Osborne  and  L.  B.  Mendel  481 


case  the  alcoholic  solut  ion  to  which  picric  acid  had  boon  added  was 
divided  into  two  parts,  one  of  which  was  allowed  to  evaporate 
slowly  until  nearly  dry.  The  semicrystalline  residue  was  extracted 
with  alcohol.  The  insoluble  residue  when  recrystallized  gave  0.2 
gram  of  lysine  picrate.  The  other  half  of  the  solution  was  neutral- 
ized with  acetic  acid  and  allowed  to  evaporate  slowly  until  a 
considerable  quantity  of  free  amino-acids  separated.  These  were 
filtered  out  and  washed  with  alcohol.  The  alcoholic  filtrate  was 
neutralized  with  sodium  hydroxide  and  treated  with  spdium 
picrate,  whereupon  a  small  precipitate  of  lysine"  picrate  formed, 
which  when  recrystallized  weighed  0.23  gram.  We  thus  obtained 
0.43  gram  of  lysine  picrate  from  100  grams  of  gliadin  correspond- 
ing to  0.15  per  cent  of  lysine  in  this  preparation. 

Whether  or  not  this  represents  all  of  the  lysine  in  this  gliadin 
cannot,  of  course,  be  determined;  but  our  previous  experience 
with  casein  has  convinced  us  that  it  is  very  difficult  to  separate 
all  of  the  lysine  picrate  from  solutions  containing  other  amino- 
acids.  Whether  the  presence  of  this  lysine  is  to  be  ascribed  to 
contamination  of  our  preparation  of  gliadin  with  other  proteins, 
or  to  the  presence  of  a  small  amount  of  lysine  in  gliadin,  is  likewise 
difficult  to  determine.  All  we  can  say  is  that  we  do  not  know  how 
any  purer  preparation  of  the  substance  heretofore  known  as 
gliadin  can  be  made,  and  our  present  opinion  is  that  future  in- 
vestigations will  show  that  gliadin  does  in  fact  yield  a  little  lysine. 

The  question  is,  therefore,  raised:  can  the  absence  of  any  amino- 
acid  from  any  protein  be  assumed  solely  because  it  cannot  be 
separated  by  direct  crystallization?  In  our  opinion  it  cannot  be 
so  assumed.  The  known  difficulty  encountered  in  trying  to  thus 
separate  all  of  any  of  the  amino-acids  from  mixtures  of  them  sup- 
ports this  view,  as  does  also  the  experience  of  Osborne  and  Jones 
and  the  more  recent  experience  of  Abderhalden.  Both  of  these 
investigations  showed  that  less  than  one-half  the  glycocoll,  alanine 
or  aspartic  acid  could  be  recovered  from  mixtures  containing  known 
quantities  of  pure  amino-acid§. 

Whether  or  not  gliadin  is  actually  deficient  in  glycocoll  or 
lysine,  we  do  know  from  incontrovertible  evidence  that  it  yields 
relatively  very  little  glycocoll,  arginine,  histidine  or  lysine  and  ex- 
tremely large  quantities  of  glutaminic  acid,  proline  and  ammonia. 
Gliadin,  therefore,  has  a  unique  constitution,  very  different  from 


482 


Gliadin  in  Nutrition 


the  tissue  proteins  of  animals,  as  well  as  from  most  of  the  other 
proteins  which  are  commonly  present  in  the  foods  of  men  and 
animals.  We  should  consequently  expect  to  find  the  value  of 
gliadin  in  nutrition  to  be  different  from  that  of  other  proteins 
which  yield  amino-acids  in  proportions  corresponding  more  closely 
with  those  obtained  from  proteins  of  animal  origin.  Accordingly 
we  have  made  a  large  number  of  prolonged  feeding  trials  on  both 
mature  and  growing  rats,  with  the  results  described  in  the 
following  pages. 

Maintenance  experiments  with  grown  rats. 

The  illustrative  protocols  which  are  presented  in  graphic  form 
are  largely  self-explanatory.  The  abscissae  of  the  curves  represent 
days  and  the  ordinates  actual  body  weight  (solid  line)  or  food- 
intake  (dotted  line)  in  grams.  In  the  charts  for  ungrown  animals 
the  average  (normal)  curve  of  growth,  plotted  from  body  weight 
data  available  for  normally  growing  animals  of  the  same  sex,  is 
represented  by  a  broken  line  for  comparison.  The  food-intake 
curve  is  plotted  from  the  weights  of  food  eaten  per  week.  Where 
numbers  are  marked  on  body  weight  curves  they  indicate  the 
time  at  which  changes  in  the  character  of  the  feeding  were  insti- 
tuted. 

In  Charts  1,  2  and  3  are  represented  the  results  of  prolonged 
maintenance  trials  with  full' grown  rats  in  which  gliadin  formed 
the  sole  nitrogenous  intake.19  These  experiments  far  exceed 
the  longest  records  of  trials  in  any  way  comparable  with  our  own 
which  have  been  reported  in  the  literature.  Henriques'  records, 
for  example,  extend  at  best  over  only  23  days.20  A  study  of  the 
dietaries  quoted  in  connection  with  these  charts  will  show  that, 
for  long  periods,  in  several  of  our  experiments,  there  was  no  possi- 
bility of  the  inclusion  of  any  other  protein  than  the  gliadin  itself 
in  the  make-up  of  the  food,  except  in  the  very  small  quantity  of 
feces  supplied  during  period  2.21    Thus  rat  130,  Chart  1,  was 

19  Some  of  the  earlier  portions  of  the  charts  in  this  paper  have  already 
been  published  elsewhere. 

20  Henriques:  Zeitschr.  f.  physiol.  Chem.,  lx,  p.  105,  1909. 

21  See  Osborne  and  Mendel:  Carnegie  Institution  of  Washington,  Publi- 
cation 156,  pt.  ii,  p.  60  for  discussion  of  the  effect  of  feces  thus  fed. 


T.  B.  Osborne  and  L.  B.  Mendel 


483 


fed  for  290  days  on  a  food  entirely  free  from  any  other  protein 
than  gliadin,  before  his  condition  became  such  as  to  render  a  change 
in  his  diet  necessary.  That  the  failure  to  be  longer  maintained 
in  a  satisfactory  condition  was  not  due  to  deficiencies  in  the  gliadin 
is  proved  by  the  rapid  recovery  of  health  and  weight  when  the 
non-protein  constituents  of  the  food  were  changed  by  replacing 
the  inorganic  constituents  and  a  part  of  the  carbohydrate  with 
" protein-free  milk."22  A  similar  condition  is  shown  by  rat  134, 
Chart  2,  but  in  this  experiment  the  decline  in  weight  occurred 
much  earlier  and  the  change  in  the  non-protein  constituents  of 
the  food  had  to  be  made  after  only  72  days.  Rat  147,  Chart  3, 
was  kept  on  the  original  gliadin  food  for  256  days,  but  at  that 
time  its  loss  of  weight  and  physical  condition  was  such  that  it 
could  only  be  restored  by  changing  the  protein  to  casein.  Later 
(see  period  6),  the  failure  to  thrive  on  the  original  gliadin  food  was 
completely  remedied  by  the  addition  of  "protein-free  milk"  to 
the  diet. 

A  possible  criticism  of  these  experiments  concerns  the  residual 
content  of  milk  protein  in  the  " protein-free  milk."  Such  analyses 
as  we  have  made  have  indicated  that  the  extent  of  this  contami- 
nation cannot  exceed  0.6  per  cent  of  the  entire  food  mixture — a 
quantity  of  "normal  protein"  far  too  small,  as  we  have  convinced 
ourselves  by  other  studies  directed  to  this  point,  to  meet  the  nutrient 
deficiency  of  gliadin  in  respect  to  growth.  However,  the  experi- 
ments which  have  been  conducted  without  the  use  of  the  protein- 
free  milk  bear  direct  testimony  in  favor  of  the  conclusion  that 
possible  traces  of  contaminating  milk  protein  cannot  in  any  way 
explain  the  satisfactory  maintenance  of  our  animals,  but  that  some 
other  substance  than  the  protein  is  the  cause  of  rapid  recovery 
induced  by  the  addition  of  the  protein-free  milk. 

In  the  light  of  such  long  continued  experiments,  extending  as 
they  do  over  a  very  considerable  portion  of  the  natural  life  of  an 
animal  whose  longevity  has  been  estimated  at  about  three  years, 
one  must  accept  these  observations  as  evidence  that,  so  far  as  main- 
tenance is  concerned,  the  protein  of  the  food  can  differ  very  widely 
in  its  amino-acid  make-up  from  the  tissue  proteins  of  the  animal 
without  affecting  the  well  being  of  the  latter. 

22  See  ibid.,  p.  80. 


484 


Gliadin  in  Nutrition 


Maintenance  experiments  with  growing  rats — failure  to  grow. 

The  following  charts  illustrate  the  inability  of  wheat  gliadin  and 
other  prolamines  to  promote  growth  under  dietary  conditions  in 
which  other  single  proteins  have  been  eminently  satisfactory. 
In  Charts  4,  5  and  6  the  curves  of  growth  with  casein,  edestin, 
and  glutenin  will  be  found  to  correspond  closely,  during  the  first 
100  days,  to  those  observed  on  animals  receiving  mixed  food. 

The  contrast  of  the  trials  with  gliadin'are  striking  in  the  extreme  ; 
and  the  results  are  the  same  if  the  alcohol:soluble  hordein  from 
barley  or  the  gliadin  from  rye  are  used.  We  have  tested  the 
gliadin  of  wheat,23  Charts  7,  8  and  9;  of  rye,23  Charts  10  and  11, 
and  the  similarly  constituted  hordein  of  barley,23  Charts  12  and  13. 
The  results  are  the  same,  whether  the  trials  be  made  at  a  very  early 
age  (compare  Chart  7)  or  somewhat  later  (compare  Chart  12). 
In  corroboration  of  the  statement  that  the  results  described  repre- 
sent true  maintenance  without  growth,  we  present  two  experi- 
ments, one  with  gelatin,  Chart  14,  and  one  with  zein,  Chart  15, 
which  show  by  contrast  the  failure  of  maintenance  when  an  abso- 
lutely inadequate  protein,  like  those  mentioned  earlier,  forms  the 
nitrogenous  constituent  of  the  food  intake. 

The  youthful  appearance  of  animals  thus  maintained  without 
growth  corresponds  in  every  respect,  so  far  as  external  characters 
go,  with  the  size  rather  than  the  age  of  the  animal.24  That  the 
failure  to  grow  is  in  nowise  attributable  to  any  toxicity  or  inhibi- 
tory property  of  the  specific  proteins  used  is  shown  by  experiments 
(see  Charts  16  and  17)  in  which  the  addition  of  a  small  proportion 
of  an  ''adequate"  protein  has  sufficed  to  induce  noteworthy 
growth.  To  determine  whether  growth  could  in  any  way  be 
induced  by  largely  increasing  the  content  of  the  "  inadequate' ' 
protein  in  the  food  mixture,  special  experiments  were  undertaken 
(see  Charts  18,  19  and  20).  The  relative  variations  in  body  weight 
in  relation  to  the  larger  protein  intake  in  the  two  series  are  too 
small  to  be  of  marked  significance. 

23  The  preparation  and  properties  of  these  proteins  are  given  by  Osborne : 
Abderhalden's  Handbuch  der  biochemischen  Arbeitsmethoden,  ii,  1909. 

24  Photographs  of  some  of  our  ungrown  animals  maintained  on  gliadin 
will  be  found  in  Osborne  and  Mendel:  Carnegie  Institution  of  Washington, 
Publication  156,  pt.  ii.  1911. 


T.  B.  Osborne  and  L.  B.  Mendel  485 


Aside  from  their  interest  in  furnishing  a  physiological  differ- 
entiation between  various  proteins,  as  exemplified  in  the  capacity 
or  failure  of  maintenance,  and  the  capacity  or  failure  of  growth, 
these  experiments  have  a  large  field  of  interest  in  presenting  a 
method  whereby  the  effective  stunting  of  animals  can  be  induced 
at  any  stage  in  the  normal  period  of  growth.  We  have  as  yet 
not  determined  the  possible  alterations  in  the  histological  make-up 
of  the  organs  and  tissues  which  may  be  correlated  with  the  suppres- 
sion of  growth.  There  is  much  in  the  recent  literature  on  infan- 
tilism which  suggests  that  the  dwarfing  may  be  secondary  to 
defects  or  alterations  in  organs,  such  as  the  ductless  glands.  One 
point  alone  may  be  emphasized  here,  namely,  that  the  capacity 
to  grow  is  by  no  means  lost  even  after  very  prolonged  periods  of 
stunting  with  the  gliadin  diet.  This  is  shown  in  Chart  7  which 
exhibits  satisfactory  growth  on  a  suitable  dietary  after  a  continuous 
suppression  of  growth  lasting  277  days,  when  the  animal  was  314 
days  old — an  age  at  which  normally  little  or  no  growth  takes 
place.25 

Gliadin  and  gestation. 

Long  continued  feeding  with  gliadin  as  the  sole  source  of  nitro- 
gen by  no  means  impairs  the  capacity  of  the  animal  to  produce 
healthy  young  and  suitably  nourish  them.  The  two  animals  whose 
records  are  presented  in  Charts  21  and  22  were 'paired  and  the 
female  (Rat  129)  gave  birth  to  a  litter  of  four  at  the  end  of 
178  days  on  the  gliadin  food  mixture.  The  young  rats  whose 
growth  records  are  reproduced  in  Charts  23,  24,  25  and  26 
were  nourished  satisfactorily  by  the  mother  during  the  first 
month  of  their  existence,  in  so  far  as  one  can  judge  by  their 
increase  in  weight,  in  comparison  with  that  of  normally  reared 
rats.  At  the  end  of  30  days,  three  rats  were  removed  from  the 
mother  and  put  upon  diets  of  casein  food,  edestin  food  and  milk 
food  respectively.  The  fourth  animal  was  allowed  to  remain  in 
the  cage  with  the  mother  whose  sole  source  of  nutriment  was  the 
original  gliadin  food  mixture.  It  will  be  noted  from  the  records 
that  whereas  the  three  removed  animals  manifested  a  normal 
growth  on  their  new  dietaries,  which  had  likewise  proved  adequate 

25  See  also  charts  cxx,  cxxi,  cxxii  and  cxxiii,  Publication  156,  pt.  ii,  Car- 
negie Institution  of  Washington. 


486 


Gliadin  in  Nutrition 


for  growth  in  many  other  instances,  the  rat  kept  with  the  mother 
began  to  evince  a  failure  to  grow  at  about  the  period  (30  days) 
when  young  rats  are  wont  to  depend  upon  extraneous  food  for 
nourishment.  In  the  present  case  this  means  that  the  young 
animal,  forced  to  depend  upon  the  gliadin  food  mixture  in  place  of 
the  milk  of  its  mother,  showed  the  typical  failure  to  grow  on  the  "inade- 
quate" diet  upon  which  the  mother  had  not  only  been  maintained  but 
had  actually  produced  young  and  secreted  milk  sufficient  in  quantity 
and  quality  to  induce  normal  growth  in  her  offspring.  No  doubt 
can  remain,  we  believe,  that  in  this  experiment,  in  which  there 
has  unquestionably  been  a  renewal,  or  new  formation,  of  body 
tissue,  very  large  in  proportion  to  the  original  weight  of  the  mother 
animal,  there  must  have  occurred  a  synthesis  not  only  of  the  "Bau- 
steine"  deficient  in  the  protein  intake,  but  likewise  of  tissue  and 
milk  components  like  the  nucleic  acids  (with  their  content  of 
purines,  pyrimidines  and  organically  combined  phosphorus), 
and  phospho-proteins,  like  casein,  etc.,  which  were  completely 
missing  in  the  special  food  intake  that  had  formed  the  sole  food  of 
the  mother  during  several  months.  Unless  one  were  prepared  to 
maintain  a  profound  alteration  in  the  chemical  make-up  of  this 
"  gliadin  family"  it  must  be  admitted  that  synthesis  in  animal 
nutrition  has  here  been  demonstrated  in  a  striking  manner. 

We  have  elsewhere26  taken  cognizance  of  the  possible  role  of 
alimentary  bacteria  in  furnishing  some  of  the  components  which 
may  be  deficient  in  the  dietary.  These  synthetic  organisms  may 
well  be  able  to  build  new  amino-acids  out  of  a  variety  of  substrates; 
and  the  possibility  is  thereby  suggested  of  the  production,  through 
bacterial  intervention,  of  complexes  missing  or  deficient  in  the 
original  food  intake.  We  can  hardly  regard  this  possibility  as  an 
explanation  of  the  ability  of  animals  to  be  maintained  on  the 
abnormal  proteins,  gliadin  and  hordein;  otherwise  there  is  no 
apparent  reason  why  they  should  not  likewise  be  maintained  by 
zein  or  gelatin,  nor  why  growth  should  not  also  be  possible  with 
every  digestible  protein  through  the  intervention  of  the  bacterial 
protein  complexes  manufactured.    It  is  more  likely  that  growth 

26  Cf.  Osborne  and  Mendel:  Carnegie  Institution  of  Washington,  Publi- 
cation 156,  pt.  ii,  p.  61,  1911. 


T.  B.  Osborne  and  L.  B.  Mendel  487 


hinges  on  the  intervention  of  some  protein  complex  not  essential 
for  the  endogenous  metabolism  of  the  individual. 

The  unique  features  of  growth  and  maintenance  on  the  special 
proteins  here  considered  serve  to  emphasize  the  fact  that  mainte- 
nance experiments  alone  cannot  suffice  to  solve  the  problem  of 
the  full  biochemical  value  of  dietaries.  Nutrition  involves  an 
ensemble  of  processes  which  are  determined  or  modified  by  factors 
whose  real  significance  is  only  beginning  to  reveal  itself.  No 
method  of  study  involving  well  controlled  conditions  need  be  cast 
aside;  but  hasty  judgment  formed  as  the  result  of  brief  feeding 
trials  on  larger  animals  containing  an  abundant  reserve  supply 
must  henceforth  be  accepted  with  extreme  caution. 


488 


Gliadin  in  Nutrition 


Chart  1,  Rat  130  9  ,  shows  long  continued  maintenance  on  a  diet  containing 
feeding  by  diseased  lungs  and  a  large  parasite,  over  40  cm.  long,  encysted  in  I 
The  diet  during  the  different  periods  is  shown  below.    During  period  2 
effect  of  this  is  discussed  in  Publication  156,  pt.  ii,  p.  61,  Carnegie  Institution  of 


PERIODS  1,  2,  AND  3. 


Gliadin  (wheat) . 

Starch  

Sucrose  

Agar....  

Salt  mixture  I.. 
Lard  


per  cent. 
....  18.0 


29.5 
17.0 
5.0 
2.5 
J28J) 
100.0 


Gliadin  (wheat).. 
Protein-free  milk. 

Starch  

Agar  

Lard  


T.  B.  Osborne  and  L.  B.  Mendel 


489 


JBo         «00        «20         ^0        460        480         500        520  J40" 

|le  protein.  The  animal's  life  was  terminated  after  546  days  of  experimental 
ur-dry  feces  from  rats  on  a  mixed  diet  was  given  each  week.    The  possible 


PERIOD  5. 

per  cent. 

•...18.0     Milk  powder   C™\ 

....  28.2  Starch    

....  20.8     Lard   " 

Efc«  5.0   

....  28.0 


60.0 
12.0 
28.0 
100.0 


100.0 


49Q 


Gliadin  in  Nutrition 


Chart  2,  Rat  134  9  ,  shows  long  continued  maintenance  on  a  diet  containing  gl 
by  an  ulcer  of  the  pylorus. 

The  diet  during  periods  1  and  2  was : 

PERIOD  1. 


Gliadin  (wheat). 

Starch  

Sucrose  

Agar  

Salt  mixture  I . . 
Lard  


T.  B.  Osborne  and  L.  B.  Mendel 


491 


500  520 


,320  340  3fo0         380         400  420         440         4fo0  480 

otein.    The  animal's  life  was  terminated  after  511  days  of  experimental  feeding 


milk. 


per  cent. 
....  18.0 
....  28.2 
....  20.8 
....  5.0 
....  28.0 
100.0 


492  Gliadin  in  Nutrition 


1 


Chart  3,  Rat  147  9 ,  shows  long  continued  maintenance  on 
lung  disease  after  445  days  of  experimental  feeding 

The  diet  during  the  different  periods  is  shown  below.  Du 
legend  on  Chart  1. 

PERIODS  1,  2,  3   AND  6. 

per  cent. 

Gliadin  (wheat)   18  0 

Starch  -  29  • 5 

Sucrose   15  0 

Agar  5.0 

Salt  mixture  1   25 

Lard  ■  •  300 

100.0 


Gliadin  (wheat) 
Protein-free  mil 
Starch  


Lard. 


T.  B.  Osborne  and  L.  B.  Mendel 


493 


280         300  320         340         3&0         380  400 


4-20  440  460 


|adin  as  the  sole  protein.  The  animal's  life  was  terminated  by 
I  quantity  of  feces  from  rats  on  a  mixed  diet  was  supplied.  See 


per  cent. 
....  18.0 
....  28.2 
. ...  20.8 

...  5.0 
. ...  28.0 

100.0 


PERIODS  5  AND  8 

per  cent. 

Casein  (cow's  milk)   18  0 

Protein-free  milk  28  2 

Starch   23  8 

Agar  

Lard  


5.0 
25.0 
100.0 


494 


Gliadin  in  Nutrition 


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T.  B.  Osborne  and  L.  B.  Mendel 


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Chart  6,  Rat  284  cf ,  shows  normal  growth  on 
a  diet  containing  glutenin  which,  together  with 
an  approximately  equal  quantity  of  gliadin. 
forms  about  80  per  cent  of  the  proteins  of  the 
wheat  kernel. 

The  diet  was: 

per  cent. 

Glutenin  (wheat)  18.0 

Protein-free  milk  28.2 

Starch  23.8 

Agar   5.0 

Lard   25  0 

100.0 


496  Gliadin  in  Nutrition 


0  DaifS  20 

Chart  7  Ra»  240  9  ,  shows  failure  to  make  more  than  slight  growth  o 
rate  after  276  days  of  stunting.    At  this  time  the  rat  was  314  days  old, 
The  diet  during  periods  1  and  2  was : 


PERIOD  1. 


Gliadin  (wheat).. . 
■  Protein-free  milk. 
Starch  


T.  B.  Osborne  and  L.  B,  Mendel  497 


300         320         340         3fc0  38o        4oo        420         440        4feO        480  SoT 

jg  gliadin  as  the  sole  protein,  and  capacity  to  resume  growth  at  a  normal 
ps  normally  grow  very  little  more. 


PERIOD  2. 


'der. 


per  cent. 
....  60.0 
....  16.0 
....  24.0 
100.0 


498 


Gliadin  in  Nutrition 


T.  B.  Osborne  and  L.  B.  Mendel  499 


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0  Oo^s  20  40  feo  go  100  120  14-0 

Chart  10,  Rat  534  9 ,  shows  failure  to  make  more  than 
slight  growth  on  a  diet  containing  gliadin  from  rye  as  its 
sole  protein.  The  animal  died  after  J52  days  of  experi- 
mental feeding  with  diseased  lungs. 

The  diet  was: 


PEiiiOi.  i. 

per  cent. 

Gliadin  (-ye)   18.0 

Gliadin  (wheat)   0.0 

Protein-free  milk   28.0 

Starch  28.0 

Lard   26.0 

100.0 


PERIOD  2. 

per  cent. 
0.0 
18.0 
28.0 
28.0 
26.0 


100.0 


/ 

/ 

1 

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li  

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+  Pro-re  ir 

i-f  ree  m 

ilk  

W 

lecitG  1  i  o 
>tein-f»-ee 

din 
n,lk> 

-  -  R^e 

0  Dai^s  20  40  fcO  80  100  120  |40  160  180  2C0 

Chart  11,  Rat  549  d%  shows  failure  to  make  more  than  slight  growth 
on  diets  containing  gliadin  from  rye  and  later  gliadin  from  wheat.  This 
experiment  was  terminated  by  the  death  of  the  rat  after  172  days  of  experi- 
mental feeding.  The  only  abnormal  condition  revealed  by  the  autopsy  was 
a  collection  of  hair  balls  in  the  stomach. 

The  die   during  periods  1  and  2  was: 

PERIOD  1.   PERIOD  2. 

per  cent  per  cent. 

Gliadin  (rye)                                                           18.0  0.0 

Gliadin  (wheat)                                                         0.0  18.0 

Protein-free  milk                                                      28.0  28  0 

Starch                                                                      28.0  26.0 

Lard                                                                       26.0  28.0 

100.0  100.0 


500 


zoo 


Chart  12;  Rat  255  9 ,  shows  failure  to  make  more  than  slight  growth  on  a  diet  con- 
ming  hordein  from  barley  as  the  sole  protein.  Hordein  is  very  much  like  gliadin  in  phys- 
il  properties  and  amino-acid  make-up  and  appears  to  have  a  similar  value  in  nutrition, 
us  rat  died  suddenly  after  249  days  of  experimental  feeding  but  no  cause  for  death  was- 
own  by  the  autopsy. 

The  diet  during  periods  1  and  2  was: 


per  cent. 

Hordein   lg  q 

Protein-free  milk   28.2 

Starch   jg  g 

Agar  

Lard  


5.0 
0.0 


100.0 


per  cent. 

Casein   jg  q 

Protein-free  milk   28  2 

Starch   18  8 

Aear   5.0 

Lard   30.0 


100.0 


Chart  13,  Rat 
256  9,  shows  .fail- 
ure to  make  more 
than  slight  growth 
on  a  diet  contain- 
ing hordein  from 
barley  as  the  sole 
protein.  Hordein 
is  very  much  like 
gliadin  in  physical 
properties  and 
amino-acid  make- 
up and  appears  to 
have   a  similar 

I  ln  nutritl°n.    The  animal  died  suddenly  after  220  days  of  experimental  feeding  but 
itopsy  failed  to  show  anything  abnormal, 
^he  diet  during  periods  1  and  2  was: 


'ordeln  

tein-free  milk. 


^gar. 
iard. 


per  cent. 

  18.0 

28.2 


litarCA   18,g 


.  5.0 
.  30.0 
100.0 


PERIOD  2. 

per  cent. 

Casein   jg  q 

Pcotein-free  milk  28  2 

Starch   18  8 

Asar  '['"  5.'o 

Lard   30.0 

100.0 


SOI 


502 


Gliadin  in  Nutrition 




\2  ^ 

«._Gela 

Prote  Ir 

tm  1- 
-free  mil 

Gelol 

--  -Kfre« 
free 

m  t  Prote 
;  milk.  5<j 
lm.  Prot( 
D^-HC  ^ 

%> 

\ 

0  Do^S  20 


Chart  14,  Rat  615  cf,  shows  failure 
to  grow  or  even  be  maintained  during 
period  1  on  a  diet  containing  gelatin  as 
its  sole  protein,  and  recovery  when  one- 
half  of  the  gelatin  was  replaced  by  glia- 
din. The  final  fall  in  weight  was  due  to 
diseased  lungs  which  caused  death. 

The  diet  during  periods  1  and  2  was: 


PERIOD  1. 

per  cent. 

Gelatin   18.0 

Protein-free  milk  28.0 

Starch   27.0 

Lard   27  0 

100.0 


PERIOD  2. 

Equal  parts  of  gela- 
tin food  (as  in  period 
1)  and  gliadin  food. 

per  cent. 

Gliadin   18.0 

Protein-free  milk  28.0 

Starch   26.0 

Lard   28.0 

100. 0 


4  

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Chart  15,  Rat  634  cf,  shows  a  rapid 
decline  in  weight,  despite  a  food  intake 
quite  sufficient  for  maintenance,  when 
the  diet  contained  zein  as  its  sole  pro- 
tein. Note  the  rapid  repair  and  growth 
when  the  zein  was  replaced  by  lactal- 
bumin  and  sudden  decline  when  the 
rat  was  again  placed  on  the  zein  food. 

The  d  et  in  the  different  periods  was: 


PERIODS  1  AND  3.  PERIOD  2. 


grams. 

per  cent. 

Zein  

18.0 

Lactaibumin   18.0 

Protein-free  milk  28  0 

Protein-free  milk  28.0 

Starch  

24.0 

Starch   29.0 

30.0 

Lard   25.0 

100.0 

100.0 

Water 

 15  cc. 

T.  B.  Osborne  and  L.  B.  Mendel  503 


120 


s 

J7 

/  / 

// 

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/  1 

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1  f opd  2 

/  1 
/  / 

Gl.o< 

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n  free  m 

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/  / 

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1/ 

if 

0  Daijs   20  fcO  80  100  120 


Chart  16,  Rat  287  cf ,  shows  nor- 
mal growth  on  a  diet  in  which  the 
protein  consisted  of  1  part  casein  and 
3  parts  of  gliadin.  Note  the  effect 
on  the  rate  of  growth  induced  by 
this  small  proportion  of  casein.  Cf. 
Charts  7,  8,  10,  13,  18  and  19. 

The  diet  consisted  of  a  mixture  of 
one  part  of  the  casein  food  with  three 
parts  of  the  gliadin  food. 

^GLIADIN  POOD.  CASEIN  FOOD. 

'percent.  percent. 

Gliadin   18.0      Casein   18.0 

Protein-free  milk  28 . 2      Starch  32.5 

Starch   28.8      Sucrose   17.0 

Agar   5.0      Agar   5.0 

Lard  ^28 .0  Salt  m ixture  I... ,  2.5 

100.0      Lard  •  25.0 

100.0 


Chart  17,  Rat  280  cf ,  shows  nor- 
mal growth  on  a  diet  in  which  the 
protein  consisted  of  1  part  casein 
and  3  parts  of  gliadin.  Note  the  ef- 
fect on  the  rate  of  growth  induced 
by  this  small  proportion  of  casein. 
Cf.  Charts  7,  8,  10.  13,  18  and  19. 

The  diet  consisted  of  a  mixture  of 
one  part  of  the  casein  food  with  three 
parts  of  the  gliadin  food. 


GLIADIN  FOOD. 

per  cent. 

Gliadin   18.0 

Protein-free  milk  28.2 

Starch   28.8 

Agar   5.0 

Lard   28.0 

100.0 


CASEIN  FOOD. 

per  cent. 

Casein   18.0 

Starch   32.5 

Sucrose   17.0 

Agar   5.0 

Salt  mixture  I...  2.5 

Lard   25.0 

100.0 


0  Datjs  ZO 


—  Gh 


P.col  P-otem--f 


Chart  18,  Rat  588  9  ,  shows  fail- 
ure to  grow  on  a  diet  containing 
gliadin  as  the  sole  protein  and  an 
artificial  imitation  of  the  natural  pro- 
tein-free milk.  After  114  days  the 
artificial  protein-free  milk  was  re- 
placed by  natural,  but  the  decline  in 
weight  which  had  begun  was  not 
stopped  by  this  change.  The  autopsy 
showed  no  adequate  cause  for  death. 
The  diet  in  periods  1  and  2  was: 


ODotjS  20  44  60  80  '00  I 

PERIOD  1. 

per  cent. 

Gliadin   18.0 

Artificial  protein-free  milk.  30.0 

Starch   22.0 

Lard   30.0 

100.0 


PERIOD  2. 

per  cent. 

Gliadin   18.0 

Protein-free  milk   28  0 

Starch   26.0 

Lard  ■  28.0 

100.0 


 T 


50 


ortrflc 

■f  r 


ol  Prote 


\ 


bO 


80 


100 


Chart  19,  Rat  594  c?,  shows  failure 
to  make  more  than  slight  growth  on  a 
diet  containing  gliadin  as  its  sole  pro- 
tein and  an  artificial  imitation  of  the 
natural  protein-free  milk.  The  animal 
died  after  92  days  of  experimental  feed- 
ing. Calculi  were  found  in  the  bladder 
and  left  kidney. 

The  diet  was: 


per  cent. 

Gliadin   25.0 

Artificial  protein-free  milk   30.0 

Starch   15.0 

Lard  ■  30.0 

100.0 


/ 

/ 

/ 

/ 

/ 

/ 

/ 

/ 

 1— 

Y  

/ 

.-Glyodu 
1 

—f  

35%ror1 

f.c.al  ?t 

ot^Kfre 

e  m>  Ik 

/  . 

/^-""^ 

Bo 


Chart  20,  Rat  603  cf,  shows  failure  to 
grow  at  normal  rate  on  a  diet  containing 
gliadin  as  the  sole  protein.  In  this  ex- 
periment an  artificial  imitation  of  the 
natural  protein-free  milk  was  used.  This 
animal  died  after  105  days  of  experi- 
mental feeding  with  diseased  lungs. 

The  diet  was: 

per  cent. 

Gliadin  35.0 

Artificial  protein-free  milk   30.0 

Starch   5.0 

Lard  •  30  0 

100.0 


504 


T.  B.  Osborne  and  L.  B.  Mendel 


505 


506 


Gliadin  in  Nutrition 


sr 

1 

J* 

-Gl.od 

m  +  Pre 

fein-Fn 

:i 

\  y — \ 

c 
+- 
*> 

T3 

— td  

1 

 1  

i 

\  / 

Chart  22,  Rat  168  cf,  shows  maintenance  and  fertility  on  a  diet  containin 
gliadin  as  its  sole  protein.  After  154  days  this  rat  was  paired  with  Rat  129,  fou 
young  being  the  result  of  the  mating.  The  animal  died  with  diseased  lungs  afte 
230  days  of  experimental  feeding. 

The  diet  was: 


PERIOD  1. 

per  cent. 

Gliadin   18.0 

Protein-free  milk   28.2 

Starch   20.8 

Agar   5.0 

Lard  >.  28.0 

100  0 


PERIOD  2. 

per  ce 

Edestin   18. 

Protein-free  milk  

Starch   20. 

Agar  ••  •■•  9 

Lard  


T.  B.  Osborne  and  L.  B.  Mendel 


20  40  60 


100  120  140  160  180  200  220 


240         260         280  300 


Iflb    w  , ^ '    °WS  n°rmal  gr°Wth  during  284  days  on  a  diet  obtaining  all  of  the  ingredi- 
fclk    Note  the  vigorous  growth  of  this  animal  which  was  produced  and  suckled  period 
ther  whose  food  during  the  previous  178  days  contained  gliadin  as  its  sole  protein  ' 


period  2 


Milk  powder. 

Starch  

Lard  


per  cent. 

  60.0 

  16.0 

  24.0 

100.0 


508  Gliadin  in  Nutrition 


/ 

/ 

o  

/' 

/ 



1 

— 1 

o  

// 

-V 

0 

A 

// 
// 

o  

// 
// 
/  / 
/  / 

\-- 

V 

5   ■  

/ 

/ 

/  / 

j— 

3 

1 

1  / 

;  

/ 

-rf 

?  —  

// 
//« 

5  

/  /  / 

2/  / 

— V 

 c 

»sein  t 

Prote, 

i-free  r 

-> 

i  ■ 

<r  

0   Daus  20 


80 


180 


Chart  24,  Rat  379  d\  shows  normal  growth  on  a  diet  containing 
casein  as  its  sole  protein  up  to  an  age  of  120  days.  The  subsequent 
fall  in  weight  is  characteristic  for  animals  thus  fed  and  will  be  discussed 
in  a  later  paper.  This  animal  was  produced  and  suckled  by  a  mother 
previously  fed  for  178  days  on  a  diet  containing  gliadin  as  its  sole  pro- 
tein. 

The  food  during  period  1  was  its  mother's  milk;  during  period  2 
as  follows: 


PERIOD  2. 

per  cent. 

Casein   18 «° 

Protein-free  milk   28 • 2 

Starch   23-8 

  5.0 


Agar. 
Lard. 


25.0 

ioo.o 


T.  B.  Osborne  and  L.  B.  Mendel 


509 


Chart  25,  Rat  380  9 ,  shows  normal  growth  on  a  diet  containing  edestin 
as  its  sole  protein  up  to  an  age  of  120  days.  The  subsequent  fall  in  weight  is  charac- 
teristic for  animals  thus  fed  and  will  be  discussed  in  a  later  paper.  This  animal 
was  produced  and  suckled  by  a  mother  previously  fed  for  178  days  on  a  diet  con- 
taining gliadin  as  its  sole  protein. 

The  food  during  period  1  was  its  mother's  milk;  during  period  2  as  follows : 

PERIOD  2. 


Edestin. 


Protein-free  milk. 
Starch  


Agar. 
Lard. 


.  18.0 
.  28.2 
.  20.8 
.  5.0 
.  28.0 

100T0 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  XII,  NO.  3. 


Gliadin  in  Nutrition 


80 


/ 
— 

y 

s 

/ 

/ 

/ 

/  y 
// 

X 

/ 

¥~  

Glii 
*-  - 

dm   1-  f 
Free  rr 

Vote.n- 
ilk 

 Cos 

ein  +  P 

-otein-f 

■ee  milt 

x  

O'Datp  2o  40  fcO  80  100  120  140  'feo  1 80  200  220 


Chaet  26,  Rat  381  9  ,  shows  failure  to  grow  on  a  diet  containing  gliadin 
as  its  sole  protein,  period  2,  and  resumption  of  growth  when  the  gliadin 
of  the  food  was  replaced  by  casein,  period  3.  The  experiment  was  termi- 
nated by  the  death  of  the  animal.  Autopsy  showed  calculi  in  the  kidneys,, 
ureters  and  bladder.  This  animal  was  produced  by  a  mother  previously 
fed  for  178  days  on  a  diet  containing  gliadin  as  its  sole  protein.  During 
period  1  it  was  suckled  by  its  mother. 

The  diet  was: 


PERIOD  2. 

per  cent. 


Gliadin   18.0 

Protein-free  milk   28.2 

Starch   20.8' 

Agar   5.0 

Lard  '   28.0 


100.0 


PERIOD  3. 

per  cent. 

Casein   18.0 

Protein-free  milk   28.2 

Starch  27.0 

Lard   27.0 

100.0 


Reprinted  from  The  Journal  or  BIOLOGICAL  Chemistry,  Vol.  XI 11,  No.  2,  1912. 


MAINTENANCE  EXPERIMENTS  WITH  ISOLATED 
PROTEINS.1 

By  THOMAS  B.  OSBORNE  and  LAFAYETTE  B.  MENDEL. 

With  the  Cooperation  of  Edna  L.  Ferry. 

{From  the  Laboratory  of  the  Connecticut  Agricultural  Experiment  Station 
and  the  Sheffield  Laboratory  of  Physiological  Chemistry  in  Yale  Uni- 
versity, New  Haven,  Connecticut.) 

(Received  for  publication,  September  26,  1912.) 

In  earlier  papers  on  our  feeding  experiments  with  isolated  food 
substances2  we  have  attempted  to  justify  the  selection  of  the  white 
rat  for  the  study  of  some  of  the  problems  connected  with  nutri- 
tion. This  animal  is  easily  reared  and  cared  for.  Its  small  size 
reduces  the  food  requirement  to  a  magnitude  which  falls  within 
the  range  of  experimental  possibility  where  the  preparation  of 
special  dietaries  by  laborious  processes  is  a  fundamental  prerequi- 
site. A  possible  advantage  in  the  use  of  smaller  animals  like  those 
which  we  have  selected  lies  in  the  fact  that  marked  changes 
in  nutritive  equilibrium  speedily  manifest  themselves.  Further- 
more, the  longevity  of  this  animal  is,  according  to  Donaldson, 
about  three  years;  so  that  the  first  year  of  life  corresponds  to  a 
long  span  in  terms  of  human  years.  Not  insignificant  is  the  addi- 
tional fact  that  the  white  rat  has  in  recent  years  been  made  the 
subject  of  exceptionally  extensive  measurements  in  respect  to 
growth  and  various  features  of  development  at  the  Wistar  Insti- 
tute in  Philadelphia  and  elsewhere.  In  this  way  physical  standards, 
so  to  speak,  have  been  established  for  this  animal.  Inasmuch  as 
we  have  successfully  maintained  albino  rats  over  periods  of  more 

1  The  expenses  of  this  investigation  were  shared  by  the  Connecticut 
Agricultural  Experiment  Station  and  the  Carnegie  Institution  of  Wash- 
ington, D.  C. 

2  Osborne  and  Mendel :  Feeding  Experiments  with  Isolated  Food  Sub- 
stances. Carnegie  Institution  of  Washington,  Publication  156,  Parts  I 
and  II,  1911;  Zeitschr.  /.  physiol.  Chem.,  lxxx,  p.  307,  1912;  this  Journal, 
xii,  p.  473,  1912. 

233 


234  Maintenance  with  Isolated  Proteins 

than  600  days  on  artificially  prepared  food  mixtures  we  believe 
that  the  adaptability  of  the  animal  for  the  purposes  under  consid- 
eration must  *be  admitted.  The  chief  obstacles  to  success  have 
been  those  diseases,  usually  involving  the  lungs,  which  other  work- 
ers with  rats  have  also  found  troublesome.  These  diseases,  how- 
ever, have  made  less  inroad  upon  our  carefully  selected  and  cared 
for  experimental  animals  than  upon  the  animals  of  our  stock 
colony.  We  believe  that  by  the  application  of  further  hygienic 
precaution  the  disease  factor  can  be  largely  eliminated. 

The  work  of  earlier  investigators,  who  have  attempted  to  deter- 
mine whether  any  single  protein  or  combination  of  proteins  is 
capable  of  supplying  the  nitrogenous  needs  of  the  body,  has  been 
reviewed  in  an  earlier  publication.3  If  we  except  the  investiga- 
tions of  Rohmann,4  the  details  of  which  have  not  yet  been  pub- 
lished, it  would  appear  from  the  records  of  our  predecessors  that 
"  experiments  in  which  one  form  of  protein  has  been  given  as  the 
sole  source  of  nitrogen  for  a  long  period  demonstrate  that,  in  spite 
of  the  abundance  of  nitrogen  in  the  diet,  the  animal  ceases  to 
thrive.5  Experiments  in  which  the  foodstuffs  administered  were 
sufficiently  "pure"  to  give  some  permanent  significance  to  the 
results  obtained  are  few  in  number.  Occasional  records  show 
that  animals  have  been  kept  over  one  hundred  days;  as  a  rule, 
however,  part  of  this  period  has  been  characterized  by  a  decline 
in  body  weight  which  obviously  must  vitiate  the  success  of  any 
demonstration  of  maintenance  on  "  artificial"  diets.  The  reserve 
store  of  nutrients  in  many  animals  is  sufficient  to  keep  them  alive 
for  considerable  periods  even  on  inadequate  dietaries,  so  that  the 
continuance  of  life  is  by  no  means  equivalent  to  adequate  nutri- 
tive maintenance.  This  fact  has  too  often  been  overlooked  in 
the  interpretation  of  nutrition  experiments. 

The  futility  of  studying  nutrition  by  methods  in  which  even  the 
"  control"  animals  decline  ought  to  be  obvious  and  is  brought  to 
mind  by  recent  experiments  of  Frank  and  Schittenhelm6  who  failed 

3  Osborne  and  Mendel;  Carnegie  Institution  of  Washington,  Publication 
156,  Part  I,  p.  6,  1911;  see  also  Cathcart:  The  Physiology  of  Protein  Metabo- 
lism, 1912,  p.  74. 

4  Rohmann:  Biochem.  Zeitschr.,  xxxix,  p.  507,  1912. 

5  Cathcart :  The  Physiology  of  Protein  Metabolism,  1912,  p.  74. 

6  Frank  and  Schittenhelm:  Therapeutische  Monatshefte,  xxvi,  p.  112,  1912. 


T.  B.  Osborne  and  L.  B.  Mendel  235 

to  nourish  rats  adequately  on  a  diet  of  egg-white,  starch,  glucose, 
fat,  cellulose  and  salt  mixture.  Their  rats  declined  within  thirty 
days  on  this  diet  which  was  selected  to  be  compared  with  a  dietary 
in  which  completely  digested  proteins  formed  the  source  of  nitro- 
gen. The  authors  emphasize  the  fact  that  such  digestion  prod- 
ucts may  at  times  contain  objectionable  toxic  substances  (amines?). 
Their  insistence  on  the  use  of  "pure"  proteins,  however,  loses 
part  of  its  significance  in  the  light  of  the  fact  that  none  of  their 
artificial  feedings  were  reasonably  successful  even  with  supposedly 
favorable  selections  of  diet. 

In  explanation  of  the  failures  of  our  predecessors  various  sug- 
gestions have  been  offered.  The  failure  to  eat  sufficient  food  has 
clearly  been  a  frequent  obstacle,  as  has  been  emphasized  among 
others  by  McCollum.7  Aside  from  this,  however,  "much  of  the 
earlier  work  in  this  connection,"  as  Cathcart  remarks,  "was 
faulty  owing  either  to  the  manner  in  which  the  experiments  were 
carried  out,  or  to  the  fact  that  the  diets  could  not  be  regarded  as 
'pure/  i.e.,  the  protein  used  was  not  absolutely  free  from  impuri- 
ties." He  adds:  "Notwithstanding  this  it  has  been  found  that  if 
the  animals  be  kept  for  a  prolonged  period  on  one  diet  they  invari- 
ably die  in  spite  of  an  abundant  caloric  intake.8  We  propose  to  con- 
sider this  criticism  carefully  in  the  experiments  to  be  reported  below. 

The  necessity  of  long  continued  experiments  calls  for  particular 
emphasis.  Physiological  alterations  dependent  upon  the  gradual 
depletion  of  a  small  store  of  essential  tissue  material  may  mani- 
fest themselves  with  extreme  slowness;  and  the  fact  that  a  satis- 
factory nutritive  balance  can  be  maintained  for  a  week  or  two  or 
even  a  month  in  some  cases  is  no  guarantee  of  either  the  ultimate 
success  of  the  dietary  or  of  the  impossibility  of  a  decline  owing  to 
the  inappropriate  exhibition  of  an  essential  ingredient  (cf.  Charts 
1,  2  and  3).  This  has  further  been  brought  out  most  strikingly  in 
the  splendid  study  of  Hart,  McCollum;  Steenbock  and  Humphrey 
on  the  physiological  effects  on  growth  and  reproduction  of  rations 
balanced  from  restricted  sources.9  They  have  shown  that  animals 
fed  rations  from  different  plant  sources  and  comparably  balanced 

7  McCollum:  Amer.  Journ.  of  Physiol.,  xxv,  p.  120,  1909. 

8  Cathcart :  The  Physiology  of  Protein  Metabolism,  1912,  p.  74. 

9  Hart,  McCollum,  Steenbock  and  Humphrey:  University  of  Wisconsin 
Agricultural  Experiment  Station,  Research  Bulletin  No.  17,  1911. 


236  Maintenance  with  Isolated  Proteins 

in  regard  to  the  supply  of  digestible  organic  nutrients  and  pro- 
duction therms  were  not  alike  in  respect  to  general  vigor,  size  and 
strength  of  offspring  and  capacity  for  milk  secretion.  The  records 
extend  over  three  years  so  that  the  gradual  manifestations  of  de- 
parture from  the  normal  standards  of  health  and  physiological 
efficiency  which  failed  to  reveal  themselves  in  the  earlier  period 
were  not  overlooked.  As  these  authors  say,  "  unquestionably  the 
physiological  value  of  a  ration  is  largely  dependent  upon  its  chem- 
ical constituents,  but  the  usual  determinations  made  on  feeding 
materials  do  not  reveal  the  character  or  manner  of  combination 
of  many  of  the  constituents.  Consequently  the  physiological 
value  can  be  determined,  in  the  present  state  of  our  knowledge, 
only  by  long  continued  observations  of  the  reaction  of  the  feed  on 
the  animal." 

A  consideration  of  the  prolonged  maintenance  of  any  animal 
raises  the  question  as  to  its  natural  duration  of  life.  In  the  case 
of  the  white  rat  we  know  of  few  statements  which  can  lay  claim 
to  any  accurate  basis.  The  current  view,  based  apparently  on  a 
statement  by  Donaldson,  that  the  average  life  span  of  the  albino 
rat  is  about  three  years,  rests  on  the  fact  that  there  had  been  no 
records  of  a  survival  of  this  period  in  animals  under  observation. 
Recent  studies  by  Slonaker10  indicate  that  a  white  rat  can  live  to 
an  age  of  1361  days.  The  incidence  of  disease  in  a  large  rat 
colony  must  be  taken  into  account  in  connection  with  duration 
tests;  and  it  has  proved  to  be  less  detrimental  among  our  isolated 
experimental  animals  than  in  the  larger  colony  where  the  animals 
are  maintained  on  mixed  food  under  conditions  more  nearly  like 
those  to  which  they  are  accustomed  by  nature.  In  fact  the  mor- 
tality among  our  experimental  rats  has  thus  far  been  smaller  than 
among  our  supply  animals. 

The  weight  of  the  animals  constitutes,  we  believe,  the  best 
index  at  the  present  time  of  satisfactory  nutritive  maintenance. 
The  necessity  of  distinguishing  carefully  between  nutrition  in 
maintenance  and  in  growth  has  been  emphasized  in  another  place.11 

10  Slonaker:  Journal  of  Animal  Behavior,  ii,  pp.  20-42,  1912;  also  The 
Effect  of  a  Strictly  Vegetable  Diet  on  the  Spontaneous  Activity,  the  Rate 
of  Growth,  and  the  Longevity  of  the  Albino  Rat,  Leland  Stanford  Junior 
University  Publications,  1912. 

11  Osborne  and  Mendel:  Zeitschr.  f.  physiol.  Chem.,  lxxx,  p.  307,  1912. 


T.  B.  Osborne  and  L.  B.  Mendel  237 


Perhaps,  as  Waters  has  suggested,  the  term  maintenance  has  been 
used  somewhat  loosely  in  the  past;  but  like  others  we  have  been  in 
the  habit  of  regarding  the  animal  in  maintenance  when  its  live 
weight  was  constant.  "A  more  correct  definition  of  the  term 
would  perhaps  be  to  say  that  an  animal  was  in  maintenance  when 
its  body  was  in  energy  balance,  but  the  live  weight  has  been  the 
conventional  measure  of  our  maintenance  values. "l2  In  the  pres- 
ent report  we  are  concerned  solely  with  the  maintenance  features. 

Under  selected  conditions  of  diet  an  animal  can  be  maintained 
adequately  without  growth  (cf.  Chart  4).  Furthermore  a  repara- 
tion of  tissue  is  not  necessarily  identical  with  growth  (cf.  the 
weight  gained  by  Rat  134,  in  Chart  5,  days  72  to  120,  which  shows 
that  it  is  possible  to  restore  tissue  loss,  exemplified  in  decline  of 
body  weight,  by  the  use  of  dietaries  which  are  inadequate  for 
growth).  Recovery  from  the  decline  due  to  malnutrition,  for 
example,  may  thus  be  brought  about  by  gliadin  feeding.  The 
possible  dissimilarity  of  the  processes  of  maintenance,  growth, 
and  repair,  in  so  far  as  they  affect  the  role  of  proteins  in  nutrition, 
has  also  been  emphasized  by  McCollum.13  The  distinctions  here 
made  are  illustrated  by  many  of  the  charts  in  the  appendix. 

It  is  perhaps  unnecessary  to  remark  that  there  may  be  involved 
in  the  problems  of  maintenance  and  growth  many  other  factors, 
viz.,  the  total  energy  intake,  the  character  of  the  inorganic  salts, 
the  specific  nature  of  the  carbohydrates  in  the  diet,  quantitative 
and  qualitative  differences14  in  the  proteins  administered  as  well 
as  the  indefinable  so-called  "hormones."  Indeed  some  writers 
at  the  present  time  believe  that  in  the  latter,  as  yet  unknown 
factors,  rests  the  secret  to  nutritive  success.  Thus  Cathcart  has 
lately  written,  "it  is  clear  that  apart  from  the  caloric  intake  and 
the  protein,  carbohydrate  and  fat  content  of  the  food,  there  is 
some  factor  or  factors  which  influences  the  utilization,  perhaps 

12  Waters :  The  Capacity  of  Animals  to  grow  under  Adverse  Conditions, 
Proceedings  Society  for  the  Promotion  of  Agricultural  Science,  xxix,  p.  3, 
1908. 

13  McCollum :  Amer.  Journ.  of  Physiol.,  xxix,  p.  215, 1911. 

14  The  choice  of  a  protein  content  of  about  18  per  cent  in  our  food  mixtures 
has  been  determined  by  nutrition  trials  with  varying  concentrations  of 
protein  in  the  diet.  These  will  be  described  elsewhere.  The  proportions 
here  selected  fall  within  the  range  of  our  most  successful  experiments,  when 
the  non-protein  factors  are  otherwise  the  same. 


238  Maintenance  with  Isolated  Proteins 


also  the  amount  of  food  required.  This  evidence  practically  points 
to  the  presence  of  some  'mineral'  substance,  or  substances,  in 
normal  food  which  are  absent  in  'pure'  food."15  In  an  earlier  report 
it  was  stated  that  an  animal  had  been  fed  more  than  217  days  on  a 
diet  in  which  the  sole  protein  was  glutenin.  The  complete  record 
of  this  animal,  Rat  71,  is  given  in  Chart  6,  since  it  illustrates  a 
number  of  important  features  connected  with  our  feeding  experi- 
ments. It  will  be  noted  that  the  decline  after  300  days  of  experi- 
mental feeding  which  could  not  be  prevented  by  the  addition  of  a 
second  protein,  edestin,  to  the  diet  was  speedily  prevented  by  the 
administration  of  mixed  food  during  a  period  of  one  week.  When 
the  earlier  glutenin  food  was  again  fed  in  period  8  a  gradual  decline 
again  set  in;  this  was  however  promptly  averted,  and  striking 
reparation  made,  when  "  protein-free  milk"  was  used  to  replace 
the  inorganic  salts  and  part  of  the  carbohydrates  of  the  dietary. 
On  this  food  mixture  the  animal  continued  for  144  days  until  death 
ensued  after  531  days  of  experimental  feeding,  the  immediate  cause 
being  an  abscess  of  the  jaw  resulting  in  the  inability  of  the  animal 
to  eat.  Old  age  may  have  contributed  materially  to  the  final 
decline  of  this  rat.  A  peculiar  significance  centers  about  period 
6  on  the  curve  because  it  clearly  shows  that  the  factors  determin- 
ing successful  nutrition  were  here  involved  in  some  other  compo- 
nent of  the  dietary  than  the  protein  itself.  The  reasons  which 
have  led  to  the  use  of  the  protein-free  milk  as  an  adjuvant  of  our 
dietaries,  as  well  as  the  possible  criticisms  in  respect  to  a  minimal 
protein  content  which  may  attach  to  its  use,  have  been  presented 
in  detail  elsewhere.16 

We  have  already  called  attention  to  the  necessity  of  long 
continued  experiments  if  the  actual  sufficiency  of  a  diet  is  to  be 
determined.  A  deficiency  in  some  essential  ingredient  may  not 
make  itself  manifest  for  a  long  time,  and  even  then  be  difficult  to 
detect;  for  many  other  causes  than  defects  in  the  food  may  lead 
to  the  decline  or  death  of  animals  during  experiments  lasting  over 
many  months.    It  is  only  when  the  failure  to  be  maintained  is 

15  Cathcart:  The  Physiology  of  Protein  Metabolism,  1912,  p.  76;  cf.  also 
Hopkins:  Journ.  of  Physiol.,  xliv,  p.  425,  1912;  Suzuki,  Shimamura  and 
Odake:  Biochem.  Zeitschr.,  xliii,  p.  89,  1912. 

16  Osborne  and  Mendel :  Carnegie  Institution  of  Washington,  Publication 
156,  Part  II,  1911;  Zeitschr.  f.  physiol.  Chem.,  lxxx,  p.  307,  1912;  This  Journal, 
xii,  p.  483,  1912. 


T.  B.  Osborne  and  L.  B.  Mendel  239 


the  invariable  outcome  of  the  prolonged  feeding  on  a  given  diet 
that  we  are  justified  in  assuming  that  the  diet  is  in  some  way  inade- 
quate, and  then  only  when  a  prompt  and  complete  recovery 
ensues  when  the  diet  is  changed  to  one  that  is  known  to  be  in  all 
respects  sufficient  (cf.  Chart  1,  period  3;  Chart  2,  period  4;  Chart  3, 
period  5;  and  Chart  6,  period  6).  Our  experience  shows  that  every 
animal  has  sooner  or  later  declined  when  fed  with  mixtures  of  iso- 
lated and  purified  proteins,  carbohydrates  and  fats  together  with 
inorganic  matter  in  the  form  of  crystallized  salts.  In  nearly 
every  case  the  decline  has  been  sudden,  with  strong  evidence 
that  death  would  soon  have  ensued  had  not  the  food  been  changed. 
In  each  case  immediate  recovery  has  followed  a  change  in  the  diet, 
thus  showing  the  experimental  foods  to  be  inadequate  for  pro- 
longed nutrition. 

Whether  the  deficiency  of  the  purely  artificial  diet  is  to  be 
attributed  to  improper  proportions  of  its  constituents,  to  improper 
combinations  of  these  constituents,  or  to  the  lack  of  some  essential 
element,  is  at  present  difficult  to  determine.  That  the  elements 
essential  for  prolonged  maintenance  are  present  in  milk  from  which 
the  fat  and  protein  have  been  removed  is  also  shown  in  the  charts 
already  referred  to.  In  every  case  the  substitution  of  a  food 
containing  its  inorganic  constituents  and  a  part  of  its  carbohydrate 
in  the  form  of  this  so-called  "  protein-free  milk"  has  resulted  in 
immediate  recovery  of  the  depleted  animal,  and  thereafter  the 
animal  has  continued  in  a  well-nourished  state  until  its  life  ter- 
minated from  disease  or  old  age.  If  we  compare  the  body  weight 
curves  of  mature  animals  maintained  in  nutritive  equilibrium  on 
the  purely  artificial  diet  with  those  of  animals  fed  on  a  diet  known 
to  be  deficient  in  some  element  supposed  to  be  essential  for  main- 
tenance, we  find  a  marked  difference.  Thus  animals  kept  on  a 
diet  free  from  inorganic  salts  or  on  one  containing  as  its  sole  pro- 
tein either  zein  or  gelatin,  which  lack  tryptophane,  immediately 
decline  and  continue  to  fall  in  weight  throughout  the  entire  time 
of  such  feeding;  whereas  in  the  experiments  with  the  purely 
artificial  mixtures  containing  adequate  proteins  and  inorganic 
salts  a  decline  in  weight  occurs  only  after  some  time,  and  is  then, 
in  nearly  every  case,  sudden  and  severe.  That  no  serious  damage 
has  been  done  to  the  animal  is  shown  by  its  rapid  recovery  when 
protein-free  milk  is  added  to  the  food;  however,  that  it  has  suffered 
in  some  way  is  indicated  by  the  fact  that  when  restored  to  its 


240  Maintenance  with  Isolated  Proteins 


original  weight  it  cannot  then  be  again  maintained  for  any  con- 
siderable time  when  returned  to  the  original  diet. 

Whether  or  not  this  means  that  its  cells  have  lost  something 
essential  for  their  normal  activity,  which  is  furnished  by  the  "  pro- 
tein-free milk,"  cannot  now  be  determined;  but  the  experimen- 
tal evidence  is  sufficient  to  justify  a  search  for  such  a  substance  in 
milk.  Recently  published  investigations  by  Hopkins17  indicate 
that  milk,  as  well  as  other  natural  food  materials,  contains  a  sub- 
stance or  substances  which,  even  in  very  small  quantities,  suffices 
to  induce  normal  and  continued  growth,  for  several  weeks  at  least, 
in  rats  maintained  on  artificial  mixtures  of  food  substances  which 
are  otherwise  inadequate  for  growth.18 

Charts  5,  7,  8,  9,  10  and  11  show  that  with  the  aid  of  "  protein- 
free  milk"  it  is  possible  to  maintain  rats  for  periods  equal  to  practi- 
cally their  entire  adult  lives  on  foods  containing  a  single  purified 
protein,  and  also  that  the  successful  food,  proteins  may  differ 
very  widely  in  their  chemical  make-up  without  affecting  the  phy- 
sical well  being  of  the  animal  to  any  noticeable  extent.  If  we  con- 
sider that  after  hydrolysis  with  acids  gliadin  yields  25  per  cent  of 
its  nitrogen  as  ammonia,  while  casein  and  edestin  yield  only  10 
per  cent,  and  that  gliadin  yields  only  5.8  per  cent  of  its  nitrogen 
as  basic  amino-acids  while  casein  yields  22.2  per  cent  and  edestin 
31.4  per  cent,  it  is  evident  that  the  form  in  which  the  nitrogen  is 
presented  to  the  animal  is  very  different  for  each  of  these  proteins. 
These  differences  are  further  emphasized  by  the  proportion  of 
some  of  the  amino-acids  determined  by  methods  which  give  results 
of  sufficient  accuracy  to  justify  comparison. 


CASEIN 

EDESTIN 

GLIADIN 

Arginine  

3.81 

14.17 

3.16 

Histidine  \ 

2.50 

2.19 

1.56 

Lysine  

5.95 

1.65 

Glutaminic  acid  

15.55 

18.74 

43.66 

Glycocoll  

0.00 

3.80 

0.00 

*  Regarding  the  presence  of  lysine  in  gliadin,  see  Osborne  and  Mendel:  This  Journal,  xii, 
p.  480,  1912. 


17  Hopkins:  Journ.  of  Physiol.,  xliv,  p.  425,  1912. 

18  Cf.  also  Suzuki,  Shimamura  and  Odake:  Biochem.  Zeitschr.,  xliii,  p. 
89,  1912. 


T.  B.  Osborne  and  L.  B.  Mendel  241 


Another  important  difference,  which  has  usually  been  over- 
looked when  comparing  casein  with  other  proteins,  is  that  only 
about  0.1  per  cent  of  sulphur  can  be  obtained  as  sulphide  from 
casein,  whereas  from  edestin  0.«i5  and  from  gliadin  0.62  per  cent  « 
can  be  obtained,  thus  showing  that  casein  contains  very  little 
cystine. 

The  possibility  of  successful  maintenance  is  not  by  any  means 
restricted  to  the  particular  proteins  employed  in  these  experiments. 
It  happens,  however,  that  our  longest  records  involve  those  pro- 
teins which  earliest  elicited  our  experimental  interest,  and  which 
could  be  prepared  in  pure  form  most  advantageously. 

Special  importance  centers  in  the  remarkably  successful  main- 
tenance of  rats  upon  gliadin.  The  continuance  of  Rat  130,  for 
example  (see  Chart  11),  during  more  than  530  days  of  adult  life 
on  a  mixture  of  isolated  food  substances  containing  a  single  pro- 
tein deficient  in  two  familiar  Bausteine,  lysine  and  glycocoll,19 
and  which  affords  an  inadequate  diet  for  satisfactory  growth, 
furnishes  by  far  the  longest  experiment  on  record  of  "artificial" 
nutrition  and  should  serve  to  justify  the  renewal  of  studies  with 
the  isolated  foodstuffs.  We  have  at  present  records  of  twelve 
rats  which  have  been  maintained  more  than  400  days  on  compar- 
able food  mixtures  and  five  animals  whose  maintenance  record 
exceeds  500  days.  These  prolonged  nutrition  trials  exceed  in 
duration  the  best  maintenance  records  which  we  have  thus  far 
obtained  in  our  stock  colony  of  animals  fed  on  mixed  foods.  Bear- 
ing in  mind  that  these  diets  are,  in  addition  to  their  probable 
freedom  from  glycocoll  in  the  case  of  casein,  and  their  deficiency 
in  glycocoll  and  lysine  in  the  case  of  gliadin,  devoid  of  more  than 
the  merest  possible  traces  of  purines  and  of  phosphoproteins, 
except  in  the  case  of  casein,  the  synthetic  activities  of  the  organism 
are  again  clearly  brought  to  mind.  It  would  surely  be  an  extreme 
exaggeration  of  every  metabolic  probability  to  assume  that  in 
periods  extending  far  longer  than  a  year,  i.e.,  approximately 
one-half  of  an  animal's  span  of  life,  the  organism  had  conserved  its 
store  of  the  missing  chemical  complexes  or  altered  its  chemical 
make-up.  The  latter  assumption  indeed  is  completely  at  vari- 
ance both  with  the  available  evidence  regarding  tne  chemical 

19  Regarding  the  possible  presence  of  lysine  and  glycocoll  in  gliadin,  see 
Osborne  and  Mendel:  This  Journal,  xii,  p.  480,  1912. 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  XIII,  NO.  2 


242  Maintenance  with  Isolated  Proteins 


fixity  of  the  tissues  and  biological  considerations  respecting  the 
identity  of  species. 

The  possible  criticism  which  may  be  evoked  by  the  use  of  "  pro- 
tein-free milk"  containing  a  minute  trace  of  unremoved  milk 
protein  (equivalent  to  about  0.5  per  cent  of  the  entire  food), 
can  be  met  satisfactorily  only  by  maintenance  experiments  which 
we  are  now  conducting  with  artificial  mixtures  of  inorganic  salts 
and  carbohydrates.20  The  trials  have  as  yet  continued  over  too 
short  periods  to  be  in  any  way  comparable  with  the  longer  records 
above.  A  few  charts  are,  however,  appended  (see  Charts  12  and 
13)  because  they  already  indicate  a  considerable  degree  of  success 
in  the  absence  of  milk  protein  as  well  as  of  the  hypothetical  organic 
" hormones,"  etc.,  present  in  the  milk.  The  experiences  with  the 
artificial  salt  mixture  I  (see  Charts  2,  7,  10  and  14)  afford  evi- 
dence on  this  point,  for  in  these  experiments  the  animals  were 
maintained  in  good  health  for  190,  360,  400  and  200  days  respec- 
tively before  declining  in  weight. 

As  a  corollary,  so  to  speak,  to  the  preceding  experiments  in 
successful  maintenance  are  appended  a  few  records  of  failures  of 
maintenance  associated  with  the  administration  of  manifestly 
inadequate  proteins  (cf.  Charts  15,  16,  17,  18,  19,  20  and  21). 
In  several  of  these  the  ready  restoration  of  body  weight  by  the 
addition  of,  or  replacement  by,  adequate  protein  is  clearly  indi- 
cated. It  is  interesting  to  note  the  more  or  less  successful  repair 
of  nutritive  failure  by  gliadin  (see  Charts  17,  18  and  21)  which  is 
inadequate  to  promote  true  growth.21 

The  failure  to  be  maintained  on  the  inadequate  proteins,  zein 
and  gelatin,  is  not  to  be  ascribed  to  a  failure  in  utilization;  for 
nitrogen  determinations  made  on  the  feces  showed  that  the  utili- 
zation of  the  protein  was  good. 

DISCUSSION. 

The  currently  discussed  theories  of  metabolism  emphasize  the 
importance  of  the  "Bausteine"  as  fundamental  factors  in  protein 
metabolism;  and  they  lay  stress  by  inference,  if  not  by  experiment, 

■+ 

20  Osborne  and  Mendel:  Proc.  Soc.for  Exp.  Biol,  and  M?d.,  ix,  p.  72,  1912* 

21  The  physiological  role  of  gliadin  is  discussed  by  Osborne  and  Mendel: 
This  Journal,  xii,  p.  473,  1912. 


T.  B.  Osborne  and  L.  B.  Mendel  243 


upon  the  results  of  supplying  these  in  adequate  proportions  as  a 
primary  requisite.22  Now  and  then  there  has  arisen  a  protest 
against  the  general  view  that  the  similarity  of  the  molecule  of  the 
food  protein  to  that  of  the  specific  body  proteins  determines  its 
relative  food  value  to  the  animal,  and  that  any  one  of  the  essential 
cleavage  products  which  is  present  in  smallest  amount  in  the  food 
protein  determines  the  value  of  the  entire  molecule  to  the  animal. 
McCollum,23  for  example,  has  presented  experimental  data  which 
do  not  harmonize  entirely  with  the  most  widely  accepted  theories 
concerning  the  chemistry  of  protein  metabolism  and  which  he  inter- 
prets to  indicate  that  the  processes  of  cellular  catabolism  and  repair 
do  not  involve  the  construction  and  resynthesis  of  the  entire  pro- 
tein molecule.  Abderhalden  has  lately  summarized  his  view  of 
the  situation  in  these  words : 

Hier  mtissen  wir  allerdings  eine  zurzeit  noch  grosse  Liicke  in  unseren 
Kenntnissen  besonders  hervorheben.  Wir  wissen  noch  sehr  wenig  iiber 
die  Fahigkeiten  der  tierischen  Zellen  Aminosauren  zu  bilden.  Nur  fur 
Glykokoll  ist  bewiesen,  dass  es  aufgebaut  werden  kann.  Ferner  wissen 
wir  von  Tryptophan,  dass  es  offenbar  von  den  Korperzellen  nicht  gebildet 
wird.    Ebenso  scheinen  die  aromatischen  Bausteine  nicht  ersetzbar  zu 

sein  Es  muss  somit  der  Moglichkeit  gerechnet  werden,  dass 

die  Zelle  imstande  ist,  manchen  Baustein  zu  erganzen  und  damit  das  zu 
Gebote  stehende  Aminosauregemisch  besser  verwertbar  zu  machen.  Durch 
die  Hervorhebung  dieser  Liicken  in  unseren  Kenntnissen  iiber  den  inter- 
mediaren  Zellstoffwechsel  wird  das  Hypothetische  in  unseren  Vorstel- 
lungen  iiber  den  Ablauf  des  Eiweissstoffwechsels  im  tierischen  Organismus 
mit  Absicht  besonders  betont.24 

The  experimental  records  presented  in  the  preceding  pages 
suggest  that  we  have  in  the  past  greatly  underestimated  the  possi- 
bility of  a  transmutation  or  synthesis  of  amino-acids  in  the  organ- 
ism, and  that  these  chemical  processes  may  play  a  more  signifi- 
cant part  in  nutrition  than  has  been  credited  to  them  hitherto. 
The  realization  of  the  possibility  of  a  transmutation  of  amino-acids 
has  an  obvious  bearing  upon  the  question  of  the  quantity  of  pro- 
tein in  the  diet;  for  the  arguments  in  favor  of  a  liberal  protein 

22  Cf.  Mendel:  Ergeb.  d.  Physiol.,  xi,  p.  418,  1911;  Cathcart:  The  Physi- 
ology of  Protein  Metabolism,  1912. 

23  McCollum:  Amer.  Journ.  of  Physiol.,  xxix,  p.  215,  1911. 

24  Abderhalden:  Synthese  der  Zellbausteine  in  Pfianze  und  Tier,  Berlin, 
1912. 


244  Maintenance  with  Isolated  Proteins 


intake  have  in  part  been  based  upon  the  belief  that  all  of  the  Baus- 
teine  must  be  supplied  in  adequate  amounts.  The  ability  to  be 
maintained  over  long  periods  on  the  chemically  unique  protein 
gliadin  is  an  excellent  illustration  of  the  point  raised. 

To  what  extent  the  daily  "wear  and  tear"  of  metabolism  may 
require  a  new  supply  of  Bausteine  to  replace  depleted  tissue, 
remains  a  matter  of  conjecture  at  present.  Studies  to  determine 
the  amount  of  nitrogenous  material  involved  in  what  Rubner  has 
termed  the  "  Abnutzungsquote"  show  that  it  is  not  large  in  quan- 
tity. The  experiments  here  recorded  give  no  answer  to  the  possi- 
ble extent  of  the  synthesis  of  new  amino-acids  except  perhaps  in 
the  case  of  glycocoll  and  lysine.  Further  investigations  with  the 
various  proteins  must  be  undertaken  to  determine  the  relative 
minimum  quantities  of  each  of  them  essential  for  long  continued 
maintenance.  Our  foods  ordinarily  contained  18  per  cent  of 
protein — a  proportion  of  the  total  intake  found  in  the  case  of 
casein  to  be  larger  than  the  necessary  minimum  of  about  6-7 
per  cent  of  the  food.  Similar  experiments  are  now  in  progress 
with  gliadin  and  edestin. 

That  a  protein  as  unlike  the  tissue  proteins  as  is  gliadin  can, 
in  fact,  serve  for  the  construction  of  new  tissues  through  the  inter- 
vention of  the  metabolic  processes  of  the  mature  animal  is  no 
longer  to  be  doubted  in  view  of  an  experiment  reported  in  another 
paper,25  in  which  a  pair  of  rats  maintained  178  days  on  gliadin 
as  the  sole  protein  in  the  diet  produced  four  healthy  young  and 
successfully  reared  them.  This  involved  not  only  the  construc- 
tion of  the  tissues  of  the  young,  but  also  the  production  of  the 
milk  by  which  they  were  successfully  nourished. 

The  recent  observation  of  Grafe  and  Schlapfer  on  the  protein- 
sparing  action  of  organic  ammonium  salts,26  as  well  as  those  of 
Abderhalden,27  taken  in  connection  with  the  striking  experiments 
of  Knoop  and  Embden  on  the  synthesis  of  individual  amino-acids 
under  purely  experimental  conditions,  brings  the  synthetic  powers 
of  the  animal  organism  into  new  prominence.  Indeed  as  Grafe 
remarks: 

25  Osborne  and  Mendel :  This  Journal,  xii,  p.  473,  1912. 

26  Grafe  and  Schlapfer:  Zeitschr.  f.  physiol.  Chem.,  lxxvii,  p.  1,  1912; 
Grafe:  ibid.,  lxxviii,  p.  485,  1912. 

27  Abderhalden:  ibid.,  lxxviii,  p.  1,  1912. 


T.  B.  Osborne  and  L.  B.  Mendel  245 


Da  wir  hier  erst  am  Anfange  unserer  Kenntnisse  auf  einem  ganz  neuen 
Gebiete  des  intermediaren  Stoffwechsels  und  der  synthetischen  Leistungs- 
fahigkeit  des  tierischen  Organismus  steheti,  haben  zunachst  natiirlich  alle 
Deutungsversuche  nur  sehr  untergeordnete  Bedeutung  und  konnen  nur  als 
Arbeitshypothesen  dienlich  sein. 

Further  evidence  of  the  physiological  efficiency  of  animals  main- 
tained on  single  proteins  is  brought  out  by  the  fact  that  the  capac- 
ity of  reproduction  has  not  been  impaired  in  these  animals  and 
no  obvious  physical  defects  or  unusual  behavior  were  discernible. 

The  long  records  from  the  continued  use  of  unchanged  rations 
afford  further  confirmation  of  what  we  have  earlier  pointed  out, 
namely,  that  monotony  of  diet  is  not  necessarily  a  troublesome 
factor  and  is  not  of  such  importance  in  nutrition  problems  as  is 
usually  supposed.28 

To  what  extent  alimentary  bacteria  may  intervene  to  furnish 
adequate  building  stones  to  the  organism  by  utilizing  the  products 
of  digestion  found  in  the  alimentary  tract  as  their  own  nutritive 
pabulum  and  reconverting  it  by  means  of  their  eminent  synthetic 
capacity  into  products  suitable  for  the  higher  organisms  cannot  be 
definitely  stated.  It  is  not  impossible  that  in  the  case  of  such 
substances  as  ammonium  salts  they  may  render  some  effective 
service  by  converting  the  nitrogen  into  other  assimilable  forms. 
We  do  not  at  present  regard  these  bacterial  possibilities  as  an 
adequate  explanation  of  the  nutritive  success  recorded;  otherwise 
there  is  no  apparent  reason  why  a  protein  defective  for  growth 
should  not  be  rendered  efficient  through  this  means.  The  con- 
tributions of  the  microorganisms  to  nutrient  efficiency  need  further 
investigation  and  the  possibilities  have  lately  been  clearly  sum- 
marized by  Armsby.29 

We  believe  that  experiments  such  as  those  reported  in  this 
paper  show  the  possibility  of  approaching  certain  of  the  problems 
of  nutrition  by  new  and  hitherto  discredited  methods  of  study. 
The  exceptionally  long  periods  of  feeding  and  observation  involved 
in  our  successful  trials  are  in  striking  contrast  with  most  of  the 
previously  published  records.    If  we  had  been  content  to  discon- 

28  Cf.  Hart,  McCollum,  Steenbock  and  Humphrey:  University  of  Wis- 
consin, Agricultural  Experiment  Station,  Research  Bulletin  No.  17,  1911. 

29  Armsby:  The  Nutritive  Value  of  the  Non-protein  of  Feeding  Stuffs, 
Bureau  of  Animal  Industry,  Bulletin  139,  1911. 


246  Maintenance  with  Isolated  Proteins 


tinue  the  experiments  after  a  reasonable  period  many  of  the 
declines  evidently  associated  with  imperfections  in  the  dietary, 
and  readily  checked  by  a  change  in  feeding,  would  have  escaped 
attention.  Such  facts  deserve  to  be  considered  in  relation  to 
current  work  in  which  the  criteria  of  adequate  nutrition  on  unusual 
diets  such  as  amino-acid  mixtures,  etc.,  are  sought  in  the  nitrogen 
balance  of  the  animals.  None  of  the  records,  so  far  as  we  are 
aware,  extend  over  periods  even  half  as  long  as  some  of  ours  which 
ultimately  ended  in  nutritive  failure.  Nitrogen  balances  may  at 
times  prove  singularly  deceptive,  and  give  apparently  favorable 
indications  of  equilibrium  although  the  real  nutritive  status  of  the 
animal  may  be  less  promising.  This  is  brought  out  in  the  consid- 
eration of  some  of  the  animals  reported  in  an  earlier  publication.30 
Now  that  an  animal  can  be  maintained  for  long  periods  on  a 
single  protein  there  is  prospect  of  a  successful  consideration  of 
those  other  factors  in  nutrition  which  are  even  more  illusive.  The 
relative  significance  of  the  inorganic  ions,  and  the  qualitative  value 
of  the  fats  and  carbohydrates  which  supplement  the  diet  ought 
to  be  rendered  amenable  to  study.31  In  the  case  of  the  carbo- 
hydrates we  have  already  made  observations  which  suggest  that 
it  is  no  longer  justifiable  to  consider  these  foodstuffs  from  a  single 
standpoint  without  reference  to  their  structural  individuality. 
The  consideration  of  such  facts  must,  however,  be  reserved  for 
other  communications. 


30  Osborne  and  Mendel:  Carnegie  Institution  of  Washington,  Publication 
156,  Part  I,  1911. 

31  Cf.  Osborne  and  Mendel:  This  Journal,  xii,  p.  81,  1912. 


T.  B.  Osborne  and  L.  13.  Mendel  247 


APPENDIX. 

Explanation  of  the  Charts. 

The  abscissae  of  the  curves  represent  days  and  the  ordinates,  actual 
body  weight  (solid  line)  or  food-intake  (dotted  line)  in  grams.  In  some 
of  the  charts  the  average  (normal)  curve  of  growth,  plotted  from  body 
weight  data  available  for  normally  growing  animals  of  the  same  sex,  is 
represented  by  a  broken  line  for  comparison.  The  food  intake  curve  is 
plotted  from  the  quantities  of  food  eaten  per  week.  The  numbers  on  the 
body  weight  curves  indicate  the  time  at  which  changes  in  the  character 
of  the  feeding  were  instituted. 


Maintenance  with  Isolated  Proteins 


5o 


food 


fece: 


Co; 


80 


i8o 


Chart  1,  Rat  127  d\  shows  long-continued  maintenance  on  the  die. 
diet  was  given  each  week.    The  possible  effect  of  this  is  discussed  in 
minated  by  diseased  lungs  after  499  days  of  experimental  feeding. 

PERIODS  1,  2  AND  4. 

per  cent. 

Casein  (cow's  milk)   18.0  Casein  (cow's 

Starch   32.5  Protein-free  m1 

Sucrose   21.9  Starch  

Salt  mixture  1   2.0  Agar  


Lard. 


25.0  Lard. 


100.0 


T.  B.  Osborne  and  L.  B.  Mendel  249 


- 
- 

X 

\ 

- 

5 

od- 

Casem  t 

+  p  rote  1  r 

-free  m 

ilk  

Edest. 
iroteirvfre 

■F 

xed 

 X- 

320  340 


low.  During  period  2  about  1  gram  of  air-dry  feces  from  rats  on  a  mixed 
>,  p.  61,  Carnegie  Institution  of  Washington.    The  animal's  life  was  ter- 


PERIOD  6. 

per  cent.  per  cent. 

...    18.0      Edestin  (hempseed)   18.0 

. ..    28.2      Protein-free  milk   28.0 

Starch   26.0 

  28.0 


23. 

5.0  Lard. 
25.0 


100.0 


100.0 


250         Maintenance  with  Isolated  Proteins 


100  120  140         160         180  200 


Chart  2,  Rat  133  9 ,  shows  maintenance  during  465  days  on  a  diel 
time  the  animal's  life  was  terminated  by  a  tumor  of  the  spleen.    No"  r 
ganic  salts  and  a  part  of  the  carbohydrate  of  the  original  food  mixture, 

The  diet  during  the  different  periods  is  shown  below. 


PERIODS  1  AND  3. 

per  cent. 

Edestin  (hemp  seed)   18.0 

Starch   29.5 

Sucrose   17.0 

Agar   «>.0 

  2.5 

  28.0 


Salt  mixture  I. 
Lard  


Edestin  (hempseed). 
Protein-free  milk  — 

Starch  

Agar  

Laid  


100.0 


T.  B.  Osborne  and  L.  B.  Mendel 


I 

\ 

od  - 



in  t  prof 

Jin-free 

niilk-- 

|  2 

O  0 

*■  D 
c  v 

\ 

\ 

 |€e- 

 f* 

V 

N 

i 

280         300         320         34-0         360         38o        400        420         44-0        4-60  4-80 

n  from  hempseed  formed  the  sole  protein.  At  the  end  of  that 
rery  in  periods  2  and  4  when  protein-free  milk  replaced  the  inor- 


PERIOD  5. 

per  cent.  per  cent. 

...    18. 0      Edestin  (hempseed)   22.0 

. ..    28.2      Artificial  protein-free  milk   29.5 

Starch   28.5 

  20.0 


20. 

5.0  Sucrose. 
28.0 


100.0 


100.0 


Maintenance  with  Isolated  Proteins 


Chart  3,  Rat  147  9  ,  shows  long  continued  maintenance  on  a  die"] 
lungs  after  445  days  of  experimental  feeding. 

The  diet  during  the  different  periods  is  shown  below.  During  p 
on  Chart  1. 


PERIODS  1,  2,  3  AND  6. 


per  cent. 

Gliadin  (wheat)   18.0  Gliadin  (wheat)..' 

Starch  '. . .  v  29.5  Protein-free  milk- 

Sucrose   15.0  Starch  JL 

Agar   5.0  Agar  .£ 

Salt  mixture  1   2.5  Lard   

Lard   30.0 


100.0 


T.  B.  Osborne  and  L.  B.  Mendel  253 


\ 

V 

V. 

7 

I- 

00/ 

\ 

-4)  

\ 

i 

/  c 

/Prot< 

osei  n 
:  1  n-f  ree 

+ 

Milk 

GI<Odir 

-JsU-- 

-  -Gl'.oc 

,  n  t  Pro 

e. n-f ree 

milk  \  > 

*  > 

1 
j 

is 

(j 

•/> 
0 

U 

280  300  320  340         360  380  400  420 


n  as  the  sole  protein.  The  animal's  life  was  terminated  by  diseased 
antity  of  feces  from  rats  on  a  mixed  diet  was  supplied.    See  legend 

PERIODS  5  AND  8. 


per  cent.  per  cent. 

...    18.0  Casein  (cow's  milk)   18. 0 

...    28.2  Protein-free  milk   28.2 

...    20.8  Starch   23.8 

...     6.0  Agar   5.0 

...    28.0  Lard   25.0 

100.0  100.0 


254         Maintenance  with  Isolated  Proteins 


o  

o     -  ■  ■  ■ 

o  —  — 

o  

o—  

o  

o  1  

la&'in  + 

Prote. 

i-f  ^ee 

milk- 

J 

 G 

0  Onus  20  40  60  80  100         120         140         160         ,a°         200  220 


Chart  4,  Rat  240  9 ,  shows  failure  to  make  more  than  slight  growt 
normal  rate  after  276  days  of  stunting.    At  this  time  the  rat  was  314  d 
The  diet  during  periods  1  and  2  was: 

PERIOD  1.  ' 

Gliadin  (wheat)  { 

Protein-free  milk  

Starch  

Agar  

Lard  


T.  B.  Osborne  and  L.  B.  Mendel 


300         320         34-0         3fo0  380  420         -440        460        460  500 


lining  gliadin  as  the  sole  protein,  and  capacity  to  resume  growth  at  a 
which  rats  normally  grow  very  little  more. 


PERIOD  a. 


der. 


per  cent. 
...  60.0 
...  16.0 
...  24.0 

100.0 


256         Maintenance  with  Isolated  Proteins 






u  ■ — 

— ,  

2 

— <;  

\ 

\ 

y  

<r-.  

Ct  1 1 0  d  1 

0  Daws  20  40  to  30  100  |20  140  IfcO  ISO         200         220  240 


Chart  5,  Rat  134  9  ,  shows  long  continued  maintenance  on  a  diet  conta) 
mental  feeding  by  an  ulcer  of  the  pylorus. 
The  diet  during  periods  1  and  2  was: 

PERIOD  1. 


Gliadin  (wheat) 

Starch  

Sucrose  

Agar  

Salt  mixture  I.. 
Lard  


T.  13.  Osborne  and  L.  13.  Mendel  257 




^  T 

"e,  n 

-4Vee  r> 

> 

L.  J  

320  340         3fo0  380         400         420         440         4b0  480  500  520  54S 


ie  sole  protein.    The  animal's  life  was  te  minated  after  511  days  of  experi- 


PERIOD  2. 

per  cent. 

heat)   18.0 

emilk   28.2 

  20.8 

B   50 

  28.0 


*100.0 


Maintenance  with  Isolated  Proteins 




«  Cq 

sem  t  Gl 

utenin  — 

 G 

lutemn 

/ 

 2 

7 

A- 

^- — 

\ 

4 — 

— —  

v' 

V 

1 

40        Go         do  ioo 


140        ifeo        180         2oo  2Zi 


Chart  6,  Rat  71  d%  shows  long-continued  feeding  of  isolated  foodstuffs 
mal's  life  was  terminated  after  531  days  of  experimental  feeding  by  an  abscess; 
The  diet  during  the  different  periods  was  as  follows : 


PERIOD  1. 

per  cent. 

Glutenin  (wheat)   6.0 

Casein  (cow's  milk)   12.0 

Starch  ,   24.5 

Sucrose   15.0 

Agar   5.0 

Salt  mixture  1   2.5 

Lard  •.   35.0 


PERIODS  2  AND  5. 


Glutenin  (wheat). 

Starch  

Sucrose  

Agar  

Salt  mixture  I  

Lard  


100.0 


T.  B.  Osborne  and  L.  B.  Mendel 


300         320         34o         36o  38o 


400        420        440        4£0  480 


>00         S20  54o 


j^nued  maintenance  on  glutenin  from  wheat  as  the  only  protein.  The 
nade  eating  impossible. 


PERIOD  3. 


per  cent. 

ain  (wheat)   9  0 


in  (hemp  seed) . 


ixture  I. 


9.0 
33.5 
18.5 
5.0 
2.5 
22  5 

100.0 


PERIODS  4  AND 

Mixed 


per  cent. 

Glutenin  (wheat)   18.0 

Protein-free  miik   28.2 

Starch   23  8 

Agar  

Lard  


5.0 
25.0 


100.0 


260         Maintenance  with  Isolated  Proteins 


60  80  100  120  140 


2C0         220  I 


Chart  7,  Rat  150  9  ,  shows  long-continued  maintenance  on  a  diet  conta 
days  of  experimental  feeding.  m 
The  diet  during  the  different  periods  is  shown  below.    During  period  2  a 


PERIODS  1,  2  AND  3. 


Casein  (cow's  milk)  

S  tarch  

Sucrose  

Salt  mixture  1   2  6  Lard 

Lard  


per  cent. 

..    18.0  Casein  (cow's  mlllj' 

..    32.5  Protein-free  milk.' 

..    21.9  Starch  


25.0 
100.0 


T.  B.  Osborne  and  L.  B.  Mendel 


300        22.0        340        360         380        400        420        440        460        480        500        520  540 


5  sole  protein.  The  animal's  life  was  terminated  by  diseased  lungs  after  538 
feces  from  rats  on  a  mixed  diet  was  supplied.    See  legend  on  Chart  1. 

PERIOD  5. 

Percent.  percent. 

  18.0      Milk  powder   60  0 

  28.0      Starch  WW'.'. 12  0 

  27.0      Lard  ...WW.    28  0 

  27.0 


100.0 


100.0 


262         Maintenance  with  Isolated  Proteins 


Chart  8,  Rat  141  9 ,  shows  long-continued  maintenance  on  a  diet  containing 

diseased  lungs. 

The  diet  during  the  different  periods  is  shown  below. 


PERIODS  1  AImD  4. 

per  cent. 

Casein  (cow's  milk)   18-0  Casein  (cow's  milk) 

Starch   32  5  farch 

Sucrose   219  ^crose  

Salt  mixture  I   2  6  Salt  mixture  I  

Lard   25  0  Lard 


PEBIOD  2. 


100.0 


T.  B.  Osborne  and  L.  B.  Mendel. 


263 


ein.    After  587  days  of  experimental  feeding  the  animal's  life  was  terminated  by 


AND  5, 


PERIOD  0. 

per  cent-  per  cent 

pmllk)   18.0      Milk  powder   60  0 

;Jmilk   28.0      Starch   120 

....    27.0      Lard  \\"t\\\    28  0 


27.0 


100.0 


100.0 


Maintenance  with  Isolated  Proteins 


— _ — 
<  

Casern  f 

pod  

 Co 

setn  i-  p 

cotem-fr; 

A 

/ 

\ 

/ 

i 

 Kr£ 

J 

3 

y 

V 

0  zo 


40 


60  SO  100         \Z0  140         160         180  200 


Chart  9,  Rat  139  d" ,  shows  long-continued  maintenance  on  a  die 
mental  feeding,  but  no  cause  for  death  was  shown  by  the  autopsy. 
The  diet  during  the  different  periods  is  given  below. 

PERIODS  1  AND  4. 

per  cent. 

Casein  (cow's  milk)   18.0  Casein  (cow's  milk, 

Starch   32.5  Starch  

Sucrose   21.9  Sucrose  

Salt  mixture  1   2.6  Salt  mixture  I . . 

Lard   25.0  Lard  S 


100.0 


T.  B.  Osborne  and  L.  B.  Mendel  265 


280        300  320 


340        3fe0         3&0        400        420         44<5       460  480 

gin  as  its  so'e  protein.    The  animal  died  after  468  days  of  experi- 


PERIODS  3  AND  5. 


per  cent. 

■  ■■    36.0  Casein  (cow's  milk), 

■  •    22  .5  Protein-free  milk . . . 

...    13.9  Starch  

..     2.6  Lard  

..    25  0 


per  cent. 
...  18.0 
...  28.0 
...  27.0 

. .  27.0 


100.0 


100.0 


266         Maintenance  with  Isolated  Proteins 


Chart  in  Rat  124  9  ,  shows  long-continued  maintenance  on  a  diet  containing  edestj 
609  days  of' experimental  feeding  (which  is,  so  far  as  we  are  aware  the  longest  reco 
weight    Dnring  period  2  the  rat  received  small  quanttt.es  of  feces  from  nor, 

PERIODS  1,  2  AND  3. 

per  cent. 

.  18  0  Edestin  (hem] 

Edestin  (hemp  seed)   lou  ,     „  ,  ' 

,  29  5  Artificial  proteii 

Starch   , 

....    17.0  Starch  

Sucrose   , 

5 . 0  Lard  

Aear   2  5 

Salt  mixture  I  

Lard  


100.0 


T.  B.  Osborne  and  L.  B.  Mendel 


267 


340  360 


38o        400       420       440       460       4&0  Soo 


520        540        560        560  600 


t  oh^  J?    f  >     tw   f d  uatUral  Protein-free  milk  for        "  days  out  of  the  entire 

W T  rt  ?  ?0  *5£  at  6nd  °f  tMs  Peri0d  the  animal  is  ^mewhat  above  its 
e  legend  on  Chart  1.   This  rat  is  still  at  its  original  weight  after  637  days 


per  cent. 

■  ■■    18.0  Edestin  (hempseed)  

•  ■  •    29. 5  Natural  protein-free  milk 

•  ••    24.5  Starch  

•  •■    28.0  Lard  


PERIOD  5. 


per  cent 
..  18.0 
..  28.0 
..  26.0 
..  28.0 


100.0 


100.0 


68         Maintenance  with  Isolated  Proteins 


WW 


foO 


L 

3 

i-  _  Glia 

dm  foo 

jfiodm 

food  -t- 

feces 

j^.  _G  l 

a  d 

0  D<4ms  20  4-0  60  60  100  120  i40  'CO  1 80         200         ZZ0  Z40 


Chart  11,  Rat  130  9,  shows  long  continued  maintenance  on  a  diet  cont 
mental  feeding  by  diseased  lungs  and  a  large  parasite,  over  40  cm.  long,  enc 
The  diet  during  the  different  periods  is  shown  below.    During  period  2  tl 

PERIODS  1,  2  AND  3. 

per  cent. 


Gliadin  (wheat)   18.0  Gliadin  (wheat) . . . 

Starch   29.5  Protein-free  milk..' 

Sucrose   17.0  Starch  

Agar   5.0  Agar  > 

Salt  mixture  1   2.5  Lard  

Lard   28.0 


100.0 


T.  B.  Osborne  and  L.  B.  Mendel  2 


\5 

''  \ 

'  \ 

St 

 ^ 

\ 

\ 

Gl.odm 

♦  Prote 

m-free 

trti  I  k 

Mi 

«* 

)0         32.0         340         3b0         380         400        420         440         460        480         500         SZO  .540 


I  the  sole  protein.  The  animal's  life  was  terminated  after  546  days  of  experi- 
r. 

nail  quantities  of  feces  from  normally  fed  rats.    See  legend  for  Chart  1. 

PERIOD  5. 


per  cent.  per  cent. 

...    18.0      Milk  powder   60.0 

...    28.2      Starch   12.0 

...    20.8      Lard   28.0 

...  5.0   

...    28.0  100.0 


100.0 


270         Maintenance  with  Isolated  Proteins 


200        220        2.40        260  28< 


Chart  12  Rat  271  9,  shows  long-continued  maintenance  on  a  purely  artificial  diet 
containing  casein  as  its  sole  protein.  The  animal  died,  after  277  days  of  experimental 
feeding,  with  diseased  kidneys. 

The  diet  during  the  different  periods  is  shown  below. 


PERIOD  1. 


PERIOD  2. 


Casein  (cow's  milk) . 

Starch  

Lactose. ..  i  

Agar  

Salt  mixture  I  

Lard  


per  cent. 

18.0      Casein  (cow's  milk)  

24 . 5  "  Artificial' '  protein-free  milk . 

...    24.0  Starch  

5.0  Lard  

...  2.5 


per  cent. 
...  18.0 
...  29.6 
...  20.4 
...  26.0 


26.0 


100.0 


100  0 


Chart  13,  Rat  588  9  ,  shows  maintenance 
on  a  diet  containing  gliadin  as  the  sole  protein 
and  an  artificial  imitation  of  the  natural  pro- 
tein-free milk.  After  114  days  the  artificial 
protein-free  milk  was  replaced  by  the  natural, 
but  the  decline  in  weight  which  had  begun  was 
not  stopped'  by  this  change.  The  autopsy 
showed  no  adequate  cause  for  death. 

The  diet  in  periods  1  and  2  was : 


PERIOD  1. 


Gliadin  

Artificial  protein-free 

milk  

Starch  

Lard   


per  cent. 
..  18.0 


30.0 
22.0 
30.0 


PERIOD  2. 

per  cent. 

Gliadin   18.0 

Protein-free  milk   28.0 

Starch   26.0 

Lard   28.0 

100.0 


0  De»w|S  20  °rO 


100.0 


T.  B.  Osborne  and  L.  B.  Mendel  271 


180 


20 
I 
0 


\2 

J— — 

u 
1 

-  -Gli 

id  in  -foe 

d  

--  - 

■§~JM  

-Gliadi 

ifbod  *  n 

ormal  f< 

-£  

-a 
0 

O 

v  

0  20         40  60  60         100         IZ0         140        160         l8o        200  220 


Chart  14,  Rat  142  9 ,  shows  maintenance  on  a  diet  containing 
gliadin  as  its  sole  protein.  During  periods  2  and  3  the  rat  received 
small  quantities  of  feces  from  normally  fed  rats.  For  a  discussion  of 
the  effect  of  the  sterilized  and  normal  feces  see  Publication  156,  p.  62, 
Carnegie  Institution  of  Washington.  The  animal  died  suddenly 
after  219  days  of  experimental  feeding,  but  unfortunately  no  autopsy 
was  made. 

The  diet  was  as  follows. 

PERIODS  1,  2  AND  3. 

per  cent. 


Gliadin  (wheat)   18.0 

Starch   29.5 

Sucrose   17.0 

Agar   5.0 

Salt  mixture  1   2.5 

Lard   28.0 


100.0 


272         Maintenance  with  Isolated  Proteins 


y  

— - — — — - 
\  1 

f 

\ 

V 

c 

C 

£ 

Q_ 

V 

S? 

4-  * 
£  t 

5- 

c  c 

"5  }!" 

u  0 



c 

-  s  —  -* 

0 

U 

/ 

\  _ 

 / 

60 
Dai 


PEEIOD  2. 

per  cent. 
Gelatin  food  (as  in 

period  1)   50.0 

Casein  food  (as  in 

period  4)   50  .0 


Chart  15,  Rat  477  d" ,  shows  rapid  de- 
cline on  a  diet  containing  gelatin  as  its 
sole  protein,  and  subsequent  mainte- 
nance and  repair  when  the  gelatin  was 
either  partially  or  entirely  replaced  by 
casein.  After  158  days  of  experimental 
feeding  the  animal's  life  was  terminated 
by  diseased  lungs. 

The  diet  during  the  different  periods 
is  shown  below. 

PERIOD  1. 
Gelatin  food. 

per  cent. 

Gelatin  (horn)....  18.0 
Protein-free  milk .  28.0 

Starch   27.0 

Lard   27.0 


100.0 


PERIOD  3. 

per  cent. 
Gelatin  food  (as  in 

period  1)   25.0 

Casein  food  (as  in 

period  4)   75.0 

100.0 


100.00 

PERIOD  4. 

Casein  food. 

per  cent. 

Casein  (cow's 

milk)   18.0 

Protein-free  milk.  28.0 

Starch   27.0 

Lard   27.0 


J. 

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Chart  16,  Rat  592  cf ,  shows  rapid  decline  on  a 
diet  containing  gelatin  as  its  sole  protein,  followed 
by  normal  growth  when  the  gelatin  is  replaced  by 
casein.  The  animal's  life  was  terminated  after  119 
days  of  experimental  feeding  by  calculi  in  the  bladder. 

The  diet  during  the  different  periods  is  shown  be- 
low. 

PERIOD  1.  PERIOD  2 

Gelatin  food.  Casein  food. 

per  cent.  per  cent. 

Gelatin  (horn). ...    18.0  Casein  (cow's 

Protein-free  milk   28.0  milk)   18.0 

Starch   27.0  Protein-free  milk.  28.0 

Lard   27.0  Starch   27.0 

  Lard   27.0 

100.0   

100.0 

PERIOD  3. 

per  cent. 
Gelatin  food  (as 

in  period  1). . . .  50.0 
Casein  food  (as  in 

period  2   50.0 


100.0 


T.  B.  Osborne  and  L.  B.  Mendel  273 




4 
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Chart  17,  Rat  598  9,  shows 
rapid  decline  on  a  diet  containing 
gelatin  as  its  sole  protein,  and  re- 
covery when  one-half  of  the  gela- 
tin was  replaced  by  gliadin,  a  pro- 
tein incapable  of  inducing  more 
than  very  slight  growth  when  it 
forms  the  sole  protein  constituent 
of  the  dietary.  The  animal's  life 
was  terminated  by  diseased  kid- 
neys after  120  days  of  experimen- 
tal feeding. 

The  diet  during  the  different 
periods  is  shown  below: 


PERIOD  l. 


PERIOD  2. 


per  cent. 

Gelatin  (horn)   18.0 

Protein-free  milk   28.0 

Starch   27.0 

Lard   27.0 

100.0 


per  cent. 
Gelatin  food   (as  in 

period  1)   50.0 

Gliadin  food   50.0 

100.0 


Gliadin  food. 


Gliadin  (wheat) . . 
Protein-free  milk. 

Starch  

Lard  


gram. 

.  18.0 
28.0 
26.0 
28.0 

100.0 


E  ' 

f 

£r 

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0--t- 



.  Ze. 

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liodm+prot 

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NO-* 

Chart  18,  Rat  659  9  ,  shows  rapid  de- 
cline on  a  diet  containing  zein  as  its  sole 
protein,  followed  by  recovery  when  the 
zein  was  entirely  or  partially  replaced  by 
gliadin  or  casein. 

The  diet  during  the  different  periods 
is  shown  below. 


60 


PERIOD  1. 

grams. 

Zein  (maize)   18.0 

Protein-free  milk.  28.0 

Starch   24.0 

Lard   30.0 

100.0 

Water  15  cc. 


PERIOD  2. 

per  cent. 
Gliadin  (wheat).  18.0 
Protein-free  milk  28.0 

Starch   24.0 

Lard   30.0 


PERIOD  3. 


100.0 


per  cent. 
Zein  food  (as  in 

period  1)   50.0 

Casein  food   50.0 

100.0 

Casein  food. 

grams. 

Casein   18.0 

Protein-free  milk  28.0 

'Starch   29.0 

Lard   25.0 


PERIOD  4. 

per  cent. 
Zein  food  (as  in 

period  1)   25.0 

Gliadin  food  (as 

in  period  2)....  75.0 

100.0 


100.0 


274 


Maintenance  with  Isolated  Proteins 


fv 

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ZeirY> 

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E  -S 

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 K- 

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80 


140  160 
Day 


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Chart  19,  Rat  146  a71,  shows  rapid  decline  on  a  diet  containing  zein  as  its  sole  protein, 
followed  by  speedy  recovery  when  the  zein  was  partially  or  entirely  replaced  by  casein  or 
edestin.    The  experiment  was  terminated  after  266  days  of  experimental  feeding. 

The  diet  during  the  different  periods  is  shown  below. 


PERIODS  1  AND  3. 


PERIOD  2. 


PERIOD  4. 


grams. 

per  cent. 

per  cent. 

Zein  (maize)  

18.0 

Zein  food  (as  in  period  1) 

50.0 

Zein  food  (as  in  period  1) .    50. 0 

Protein-free  milk  

28.2 

Casein  food  ({ 

is  in  period 

Edestin  food  (as  in  period 

23.8 

6)  

50.0 

5)   50.0 

Agar  

5.0 

25.0 

100.0 

100.0 

100.0 

Water  

PERIOD  5. 


per  cent. 

per  cent. 

Edestin  (hempseed) . . 

,  18.0 

Casein  (cow's  milk) . . 

18.0 

28.2 

Protein-free  milk  

.  28.2 

Starch  

20.8 

Starch  

23.8 

Agar  

5.0 

5.0 

Lard  

28.0 

25.0 

100.0 

100.0 

♦ 


T.  B.  Osborne  and  L.  B.  Mendel  275 


/ 

3 

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Vi- 

>i « 

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E 

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£ 

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 1 

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n. 

11 

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Z.e.n-i 
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 X-  =- 

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60  80 


180 


CWort 


Chart  20,  Rat  475  cf,  shows  rapid  decline  on  a  diet  containing  zein  as  its  only 
protein,  followed  by  recovery  when  one-half  of  the  zein  was  replaced  by  casein. 
Note  that  the  decline  in  period  3  when  only  one-sixth  of  the  zein  was  replaced  by 
casein  was  immediately  checked  when  3  per  cent  of  the  zein  was  replaced  by 
tryptophane. 

This  experiment  is  still  in  progress. 

The  diet  during  the  different  periods  is  shown  below: 


PERIOD  1. 


PERIOD  2. 


PERIOD  3. 


PERIOD  4. 


grams. 

Zein  (maize)   18.0 

Protein-free  milk  28.0 

Starch   24.0 

Lard   30.0 

100.0 

Water   15  cc. 


per  cent. 
Zein  food  (as  in 

period  1)   50.0 

Casein  food   50.0 

100.0 


per  cent. 
Zein  food  (as  in 

period  1)   83.33 

Casein  food  (as 

in  period  2) . . . .  16.67 

100.00 


per  cent. 

Zein  food  (as  be- 
low)  83.33 

Casein  food  (as 
in  period  2)... .  16.67 

100.00 


Casein  food. 


Zein  food. 


grams. 

Casein  (cow's 

milk)   18.0 

Protein-free  milk  28.0 

Starch   27.0 

Lard   27.0 


grams. 

Zein  (maize)   17.46 

Tryptophane   0.54 

Protein-free  milk   28 . 00 

Starch   24.00 

Lard   30.00 


100.0  100.00 
Water   15  cc. 


Maintenance  with  Isolated  Proteins 


i  , 

prdtein.free 

milk  R-ot 

jl.ad.n 
'.m-  free 

milk 

Q_     2  ^ 

E|.eS 

u 

hi 

protein- 

3 

0  20  40  60  80  100  12.0 

•Da  up 


Chart  21,  Rat  628  cf,  shows  rapid  decline  on  a  diet  con- 
taining zein  as  its  sole  protein,  followed  by  maintenance  when 
the  zein  was  replaced  by  gliadin  or  lactalbumin. 

The  diet  during  the  different  periods  is  shown  below. 

PERIOD  1.  PERIODS  2  AND  4.  PERIOD  3. 


grams. 

Zein  (maize)   18.0 

Protein-free  milk  28.0 

Starch   24.0 

Lard   30.0 

100.0 

Water   15  cc. 


per  cent. 
Gliadin  (wheat). .  18.0 
Protein-free  milk  28.0 

Starch   26.0 

Lard   28.0 

100.0 


per  cent. 
Gliadin  food  (as 

in  period  2) . . . .  75 . 0 
Lactalbumin  food   25 . 0 

100.0 


LACTALBUMIN  FOOD. 

per  cent. 

Lactalbumin 

(cow's  milk)....  18.0 
Protein-free  milk.    28. 0 

Starch  ,..  29.0 

Lard   25.0 


100.0 


Reprinted  from  The  Journal  of* Biological  Chemistry,  Vol.  XIII,  No.  I,  1912, 


A  STUDY  OF  THE  MECHANISM  OF  PHLORHIZIN 
DIABETES. 

By  FRANK  P.  UNDERHILL. 

{From,  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University. 
New  Haven,  Connecticut.) 

(Received  for  publication,  August  7,  1912.) 

The  distinctive  feature  of  phlorhizin  diabetes  in  contrast  with 
other  types  is  the  significant  diminution  in  the  blood  sugar  content. 
The  mechanism  by  which  this  condition  of  hypoglycaemia  is  estab- 
lished has  been  explained  by  a  variety  of  theories,  none  of  which, 
however,  has  received  universal  acceptance.  Of  the  numerous 
views1  that  have  been  promulgated  two  have  received  considerable 
attention.  It  was  the  opinion  of  v.  Mering2  that  the  drug  creates 
an  increased  permeability  of  the  kidney  for  sugar,  thus  leading  to 
the  passage  of  blood  sugar  into  the  urine.  To  meet  this  drain 
upon  the  sugar  of  the  blood  an  augmented  activity  of  the  blood 
sugar  regulating  mechanism  is  supposed  to  be  set  up  whereby  new 
sugar  is  formed  in  the  body  from  some  antecedent  substance,  pre- 
sumably protein.  If  this  explanation  is  correct  one  might  fairly 
assume  that  extirpation  or  ligation  of  the  kidneys  in  phlorhizin 
diabetes  by  preventing  a  renal  loss  of  sugar  would  result  in  a  restor- 
ation of  sugar  to  its  normal  percentage  in  the  blood.  Minkowski's3 
experiments  tend  ' to  support  this  position  although,  as  Levene4 
pointed  out,  Minkowski  obtained  an  increase  of  sugar  in  the  blood 
beyond  the  normal  after  extirpation  of  the  kidneys,  which  theoreti- 
cally should  not  result  if  phlorhizin  merely  has  a  specific  influence 
upon  the  kidney  and  does  not  act  upon  some  other  mechanism. 

1  For  a  discussion  of  the  problem,  cf.  Macleod:  Recent  Advances  in  Physi- 
ology and  Bio-Chemistry,  Edited  by  Leonard  Hill,  190G. 

2  v.  Mering:  Verhandl.  d.  5te  Congresses  f.  inn.  Med.,  1886,  p.  18.5. 

3  Minkowski:  Arch.  f.  exp.  Path.  u.  Pharm.,  xxxi,  p.  85,  1893. 

4  Levene:  Journ.  of  Physiol.,  xvii,  p.  259,  1894-5. 


1 6  Mechanism  of  Phlorhizin  Diabetes 


A  second  view  of  the  nature  of  phlorhizin  diabetes,  which  was 
advocated  by  Pavy  and  others,5  differs  from  the  first  primarily 
in  ascribing  to  the  kidney  the  power  of  producing  sugar.  Under 
the  influence  of  phlorhizin  the  cells  of  the  renal  tubules  are  supposed 
to  exert  a  catabolizing  action  upon  something  reaching  them  from 
the  blood,  resulting  in  the  liberation  of  dextrose  in  a  manner  com- 
parable to  that  by  which  lactose  is  set  free  by  the  cells  of  the  mam- 
mary gland. 

That  the  conditions  existing  in  pancreatic  and  phlorhizin  dia- 
betes are  similar  in  many  respects  is  well  recognized.  Thus  in 
the  starving  dog  the  D:N  ratios  obtained  in  the  two  experimental 
states  are  somewhat  alike.  Moreover,  it  is  generally  conceded 
that  in  phlorhizin  diabetes  as  well  as  in  the  condition  induced  by 
removal  of  the  pancreas  the  ability  of  the  organism  to  utilize  dex- 
trose may  be  somewhat  diminished.  The  distinguishing  difference 
between  these  two  abnormal  states  is  found  in  the  lowered  blood 
sugar  content  in  phlorhizin  diabetes  and  the  existence  of  hyper- 
glycaemia  in  pancreatic  diabetes. 

Although  numerous  investigations  have  been  carried  through 
concerning  the  relation  of  ligation  or  ablation  of  the  kidi^s  to 
phlorhizin  diabetes  a  review  of  the  literature  fails  to  reveal  an 
experiment  of  this  nature  with  animals  in  total  diabetes,  that  is, 
with  a  D:N  ratio  of  approximately  3.65.  In  the  work  of  Minkow- 
ski comparison  has  been  made  of  the  blood  sugar  content  of  dogs 
both  in  phlorhizin  and  pancreatic  diabetes  after  kidney  removal. 
He  found  that  in  pancreatic  diabetes  hyperglycaemia  was  in  evi- 
dence whereas  the  blood  sugar  content  rose  little,  if  at  all,  above 
the  normal  in  phlorhizin  diabetes.  It  appears  to  the  writer,  how- 
ever, that  the  conditions  existing  in  the  two  experimental  states 
were  too  dissimilar  for  strict  comparison.  In  pancreatic  diabetes 
a  constant  influence  is  at  work  for  one  may  reasonably  assume  that 
extirpation  of  the  pancreas  either  initiates  a  stimulus  for,  or  removes 
an  inhibition  from,  the  sugar  regulating  mechanism.  In  phlor- 
hizin diabetes,  on  the  other  hand,  to  produce  a  similar  constant 
response  on  the  part  of  the  body  the  animal  organism  must  be 
continually  supplied  with  the  necessary  quantity  of  the  drug. 
Expressed  in  other  words,  previous  experiments  -  have  been  per- 

5  Pavy,  Brodie  and  Siau:  Journ.  of  Physiol.,  xxix,  p.  467,  1903. 


Frank  P.  Underhill 


17 


formed  with  animals  in  unlike  states;  in  pancreatic  diabetes,  a 
constant  stimulus  has  been  present,  while  little  or  no  attempt  has 
been  made  to  imitate  comparable  conditions  in  phlorhizin  diabetes. 
The  production  and  maintenance  of  a  D:N  ratio  of  3.65  would 
appear  to  satisfy  the  condition  of  a  constant  stimulus.  Ablation 
or  ligation  of  the  kidneys  under  these  circumstances  would  perhaps 
constitute  an  experiment  with  conditions  more  strictly  compara- 
ble with  those  obtaining  in  pancreatic  diabetes  than  is  true  for 
many  of  the  previous  investigations.  Hitherto,  for  the  most  part, 
little  or  no  attention  has  been  devoted  to  the  question  of  phlorhizin 
dosage  or  to  the  period  of  time  which  phlorhizin  might  reasonably 
be  expected  to  exert  an  influence  upon  the  percentage  of  sugar  in 
the  blood.  At  present  it  is  well  recognized  that  the  glycosuric 
influence  of  a  single  injection  of  phlorhizin  persists,  in  dogs  at  least, 
for  a  few  hours  only.  Yet  there  are  records  of  experiments  of  the 
type  under  discussion  demonstrating  the  possibility  that  sufficient 
time  had  elapsed  since  the  last  phlorhizin  injection  for  restoration 
of  blood  sugar  content  by  utilization  of  an  excess  in  the  blood,  pro- 
vided a  high  percentage  of  sugar  in  the  blood  had  been  temporarily 
established. 

The  object  of  the  present  investigation  has  been  a  study  of  the 
changes  in  blood  sugar  content  of  animals  in  phlorhizin  diabetes 
after  ligation  of  the  kidneys  or  suppression  of  the  renal  secretory 
function.  Two  types  of  experiments  were  planned.  In  the  one, 
dogs  have  been  brought  into  a  condition  of  total  diabetes  with 
phlorhizin.  The  kidneys  were  then  ligatured  twice,  one  ligature 
being  placed  around  the  ureters  and  the  blood  vessels,  another 
designed  to  include  any  collateral  vascular  branches.  The  exper- 
iments were,  so  planned  that  the  operation  was  performed  upon 
the  kidneys  shortly  after  the  morning  administration  of  phlorhizin, 
and  a  second  injection  of  the  drug  was  given  as  usual  eight  hours 
subsequent  to  the  first.  Blood  was  analyzed  for  total  solids  and 
dextrose  content  just  previous  to  the  operation  and  at  intervals 
subsequent  to  ligature  of  the  renal  organs. 

In  the  other  type  of  experiment  the  function  of  the  kidney  as  an 
excretory  organ  was  practically  abolished  in  fasting  phlorhizinized 
rabbits  by  the  subcutaneous  administration  of 'sodium  tartrate.6 

6  Cf.  Underhill:  this  Journal,  xii,  p.  115,  1912. 


THE  JOURNAL  OF*  BIOLOGICAL  CHEMISTRY,  VOL.  XIII,  NO.  1 


18  Mechanism  of  Phlorhizin  Diabetes 

In  this  instance  the  blood  supply  to  the  kidneys  was  presumably 
uninterrupted  whereas  the  escape  of  sugar  from  the  blood  was  pre- 
vented by  the  changed  character  of  the  tubular  epithelium.  The 
establishment  of  such  a  condition  has  been  found  to  occur  after 
tartrate  injections  and  has  been  recorded  in  a  previous  paper. 
Blood  analyses  were  made  only  after  removal  of  the  kidney  function; 
for  experience  relative  to  the  blood  sugar  content  of  both  normal 
fasting  rabbits  and  phlorhizinized  rabbits  made  unessential  the 
preliminary  determination  of  the  blood  sugar  content.  By  omis- 
sion of  the  estimation  of  blood  sugar  content  before  abolition  of 
the  kidney  function  the  well-known  influence  of  the  removal  of  a 
relatively  large  quantity  of  blood  upon  the  percentage  of  sugar  in 
the  blood  was  obviated. 

The  double  ligation  of  the  kidneys  in  dogs  practically  amounted 
to  extirpation  of  these  organs  and  hence  was  equivalent  to  the 
non-participation  of  the  kidneys  in  the  sequence  of  phenomena 
following  the  operation.  In  the  case  of  rabbits,  however,  the 
circulation  through  the  kidneys  was  presumably  more  or  less  intact 
although  the  secreting  mechanism  was  abolished.7  Any  influence 
exerted  by  phlorhizin  upon  the  renal  organs  in  the  last  mentioned 
instance,  such  as  production  of  sugar,  would  therefore  be  possible 
through  the  channel  of  the  circulation.  If  the  kidney  is  specifically 
responsible  for  the  blood  su?ar  phenomena  exhibited  in  phlorhizin 
diabetes  the  results  obtained  from  examination  of  the  blood  sugar 
content  under  the  two  conditions  just  outlined  should  theoretically 
at  least  be  totally  unlike.  Ligation  of  the  kidneys  might  be  ex- 
pected under  the  experimental  conditions  to  maintain  the  con- 
dition of  hypoglycaemia  or  at  most  to  allow  blood  sugar  content 
to  become  normal.  If  the  kidneys  actually  produce  sugar  from 
some  antecedent  in  the  blood,  as  suggested  by  Pavy,  the  blood 
sugar  content  might  be  assumed  to  increase  even  above  the  normal 
by  reabsorption  in  the  absence  of  free  secretion,  provided  it  is 
granted  that  in  the  nephritis  induced  sufficient  normal  renal  cells 
are  present  to  accomplish  such  a  task.  On  the  contrary,  if  the 
renal  cells  after  tartrate  injection  are  totally  incapacitated  from 
producing  sugar  under  the  influence  of  phlorhizin,  that  is,  prac- 
tically every  cell'  has  lost  its  function — a  condition  which  is  most 

7  Underhill :  loc.  cit. 


Frank  P.  Underbill 


19 


unlikely — then  the  blood  sugar  should  behave  in  the  manner  in- 
dicated for  ligation  of  the  kidney,  that  is,  the  sugar  in  the  blood 
should  not  increase  above  the  normal.  It  has  been  found  that 
in  general  with  both  types  of  experiments  blood  sugar  content  rose 
above  the  normal. 

For  the  establishment  of  hyperglycaemia  under  either  of  the 
two  methods  outlined,  various  explanations  may  be  offered.  In 
the  first  place  it  may  be  assumed  that  phlorhizin  acts  specifically 
upon  the  kidney  rendering  this  organ  more  permeable  for  sugar, 
as  suggested  by  v.  Mering,  and  in  its  attempt  to  maintain  blood 
sugar  content  normal  the  blood  sugar  regulating  mechanism  is 
thrown  somewhat  out  of  adjustment,  the  inhibition  is  removed  or 
in  a  manner  similar  to  antibody  production  there  is  a  compensatory 
hyperfunction  and,  in  the  event  of  the  removal  of  the  kidney  func- 
tion, sugar  increases  in  the  blood  until  hyperglycaemia  obtains. 
In  the  case  of  rabbits,  from  the  standpoint  of  Pavy's  suggestion, 
hyperglycaemia  could  be  induced  by  production  of  sugar  in  the 
kidney  and  reabsorpfion  into  the  blood.  Finally,  hyperglycaemia 
may  be  explained  equally  well  on  the  assumption  that  phlorhizin 
has  a  two-fold  action :  (a)  an  influence  upon  the  kidney,  resulting 
in  augmented  permeability  for  blood  sugar  and  (b)  a  specific  activ- 
ity upon  some  other  mechanism  whereby  the  organism  continually 
produces  new  sugar  which  it  throws  into  the  blood  stream.  The 
latter  action  might  be,  however,  less  pronounced  than  the  former; 
hence,  under  ordinary  conditions  hypoglycaemia  is  found  -  asso- 
ciated with  phlorhizin  diabetes.  The  evidence  adduced  below 
points  in  this  direction. 

Methods:  Throughout  this  investigation  the  experimental 
animals  were  maintained  in  a  state  of  inanition  but  water  was 
freely  given.  With  dogs  total  phlorhizin  diabetes  was  established 
according  to  the  procedure  recommended  by  Lusk.8  For  the  oper- 
ations, which  were  performed  under  aseptic  conditions,  anaesthesia 
was  produced  by  ether  only.  After  ligation  of  the  kidneys  no ' 
anaesthetic  was  necessary  for  withdrawal  of  blood  samples  from  a 
femoral  artery.  Post-mortem  examinations  demonstrated  in 
each  case  the  complete  ligation  of  the  kidneys.  No  attempt  was 
made  in  the  experiments  with  rabbits  to  establish  a  fixed  D:N 

8  Lusk:  Amer.  Journ.  of  Physiol.,  xxii,  p.  163,  1908, 


20 


Mechanism  of  Phlorhizin  Diabetes 


ratio,  the  animals  receiving  an  injection  of  0.25  gram  phlorhizin 
subcutaneously  once  daily.  Tartrate  injections  were  also  given 
subcutaneously.  Blood  sugar  was  estimated  by  the  method  com- 
monly employed  in  this  laboratory.9  Total  solids  of  the  blood 
were  determined  in  the  usual  manner.  Glycogen  in  the  liver  was 
estimated  according  to  the  procedure  of  Pfliiger.10 

The  influence  of  ligation  of  the  kidneys  upon  the  blood  sugar  content 
of  dogs  in  phlorhizin  diabetes. 

From  the  data  presented  in  tables  1  and  2  several  points  of  inter- 
est are  indicated.  In  the  first  place  it  is  apparent  that  the  D :  N 
ratio  of  3.65  for  fasting  dogs  established  by  Lusk  was  readily 
reached  and  that  in  this  condition  hypoglycaemia  is  in  evidence. 

TABLE  1. 


Experiment  7,  Dog  X.    Bitch  of  9.5  kilos  received  three  times  daily  subcu- 
taneous injection  of  2  grams  phlorhizin. 


URINE 

BLOOD 

"  DATE 

1911 

• 

Volume 

Total  n 

Total 
Solids 

Dex- 
trose 

REMARKS 

cc. 

grams  grams 

per  cent 

per  cent 

December 

Urine  was  not  collected  un- 
til second  day  of  phlorhi- 
zin administration. 

19 

600 

9.45  36.60 

D  :  N  =  3.76. 

20 

620 

10.20  36.52 

D  :  N  =  3.58. 

21 

17  75 
15.30 

0  062 
0.172 

Blood  drawn  j  ust  before  liga- 
tion of  kidneys. 

12.00  m.  Kidneys  ligated 
four  hours  after  first  daily 
phlorhizin  injection. 

3.00  p.m.  2  grams  phlor- 
hizin as  usual. 

6.00  p.m.  Six  hours  after 
ligation  of  kidneys. 

8.00  p.m.  Animal  dead.  The 
liver  was  glycogen  free. 

9Underhill:  this  Journal,  i,  p.  113,  1905-06. 
10  Pfliiger:  Arch.  f.  d.  ges.  Physiol,  cxi,  p.  307,  1906. 


Frank  P.  Underhill 


2  I 


The  most  noteworthy  result  of  the  experiments,  however,  is  shown 
by  the  change  in  the  blood  sugar  content  of  the  blood  after  ligation 
of  the  kidneys.  It  will  be  observed  in  both  experiments  that  after 
ligation  of  the  kidneys  in  dogs  maintained  in  total  phlorhizin  diabetes 
the  percentage  of  sugar  in  the  blood  rises  above  the  normal,  although 
the  hyperglycaemia  was  much  more  marked  in  one  case  than  in 
the  other.  Accompanying  this  hyperglycaemia  is  a  significant 
diminution  in  the  percentage  of  total  solids  of  the  blood  or,  viewed 
from  the  opposite  standpoint,  an  increase  in  the  water  content. 
If  this  fact  is  taken  into  consideration  one  may  reasonably  assume 
that  the  blood  sugar  content  in  relation  to  the  other  solids  was 
actually  increased  to  an  extent  even  greater  than  the  percentage 
figures  indicate. 

Comparison  of  th*ese  figures  for  blood  sugar  content  with  those 
for  instance  of  Minkowski  obtained  in  pancreatic  diabetes  after 

TABLE  2. 


Experiment  8,  Dog  Y.   Bitch  of  11.5  kilos  received  three  times  daily 'subcuta- 
neous injection  of  2  grams  phlorhizin. 


URINE 

BLOOD 

DATE 

1911 

Volume 

Total 
Nitro- 
gen 

Dex- 
trose 

Total 
Solids 

Dex- 
trose 

REMARKS 

cc. 

grams 

grams 

per  cent 

per  cent 

December 

'Urine  was  not  collected  un- 
til second  day  of  phlor- 
hizin administration. 

19 

560 

10.53 

40.00 

D  :  N  =  3.79. 

20 

610 

10.14 

35.87 

D  :  N  =  3.53. 

21 

22.45 

21.70 
21  70 

0  087 

0  276 
0  306 

Blood   drawn   just  before 

kidney  ligation. 
10.30  a.m.    Kidneys  ligated 

2.5  hours  after  first  daily 

phlorhizin  injection. 
3.00  p.m.   2.0  grams  phlor- 
.  hizin  as  usual. 
6.00  p.m.    7.5  hours  after 

ligation  of  kidneys. 
11.00  p.m.    12.5  hours  after 

ligation  of  kidneys. 
Animal  bled  to  death.  The 

liver  was  glycogen-free. 

22  Mechanism  of  Phlorhizin  Diabetes 


kidney  extirpation,  reveals  the  interesting  fact  that  in  both  instances 
the  type  of  response  to  the  given  stimulus  is  similar;  for  in  each 
case  there  is  a  hyperglycaemia.  It  must  be  admitted,  however, 
that  in  pancreatic  diabetes  the  degree  of  hyperglycaemia  is  much 
more  marked  than  in  the  condition  induced  by  phlorhizin.  It  is 
also  of  significance  that  in  our  experiments  the  liver  failed  to  reveal 
a  trace  of  glycogen  in  the  one  hundred  grams  of  tissue  employed 
for  analysis. 

The  results  of  these  experiments  justify  the  suggestion  that  in 
phlorhizin  diabetes  there  may  be  two  types  of  action.  In  the 
first  place,  there  is  exerted  an  influence  upon  the  kidney  whereby 
this  organ  becomes  more  permeable  for  blood  sugar  and  secondly, 
there  is  evidence  of  the  stimulation  of  another  structure  or  mechan- 
ism that  functions  by  producing  sugar  or  perhaps  by  diminishing 
sugar  destruction. 

The  behavior  of  the  blood  sugar  content  in  phlorhizinized  rabbits  after 
suppression  of  kidney  secretion  by  means  of  sodium  tartrate. 

In  tables  3  to  8  inclusive  may  be  found  data  concerning  the 
percentage  of  sugar  in  the  blood  of  phlorhizinized  rabbits  after 
suppression  of  kidney  secretion  by  means  of  subcutaneous  injec- 
tions of  sodium  tartrate.  Inspection  of  these  data  will  clearly 
demonstrate  an  increased  blood  sugar  content  in  most  of  the  experi- 
ments after  exclusion  of  the  kidney  secretion  even  though  these 
animals  were  not  in  a  condition  of  total  diabetes.  It  is  apparent 
therefore  that  in  the  rabbit,  at  least,  a  condition  of  total  diabetes 
is  not  essential  for  the  production  of  hyperglycaemia  after  suppres- 
sion of  the  kidney  function. 

The  observations  recorded  here  do  not  exclude  the  possibility 
of  the  production  and  reabsorption  of  sugar  by  the  kidneys  as 
outlined  by  Pavy.  However,  in  view  of  the  fact  that  the  type  of 
response  in  this  case  coincides  exactly  with  that  observed  for  dogs 
where  the  kidneys  were  practically  removed,  it  is  probable  that 
the  mechanism  in  the  two  cases  is  similar  for  there  is  no  obvious 
reason  to  assume  that  the  character  of  reaction  is  different  in  the 
two  species  of  animals.  If  our  conception  is  correct,  then  it  is 
obvious  that  sugar  production  and  reabsorption  by  the  kidney 
can  play  only  an  insignificant  role  at  most,  since  in  the  case  of 


Frank  P.  Underhill 


23 


TABLE  3. 

Experiment  1,  Rabbit  G.    Female  rabbit  of  2400  grams  received  daily  sub- 
cutaneous injection  of  0.25  gram  phlorhizin. 


DATE 
1911 

URINE 

BLOOD 

REMARKS 

Volume 

Total 
Nitro- 
gen 

Dex- 
trose 

Total 
Solids 



Dex- 
trose 

cc. 

grams 



grams 

per  cent 

per  cent 

November 

29 

105 

0.86 

2.0 

Would  not  drink  water. 

30 

60 

1 .04 

1 .49 

Would  not  drink  water. 

December 

1 

85 

1.36 

0.98 

Animal  drank  80  cc.  water. 

2 

32 

0.072 

0.072 

Subcutaneous  injection  of  1.0 

gram  tartaric  acid,  neu- 

tralized with  Na2Co3,  dis- 

solved in  20  cc.  water. 

Drank  60  cc.  water. 

3 

3 

0.007 

trace 

Drank  30  cc.  water. 

4 

0 

18  11 

0  12 

Blood  drawn  5  hours  after 

last  phlorhizin  injection. 

The   liver  was  glycogen 

free. 

TABLE  4. 

Experiment  2,  Rabbit  H.    Male  rabbit  of  2500  grams  received  daily  subcu- 
taneous injections  of  0.25  gram  phlorhizin. 


DATE 

1911 

URINE 

BLOOD 

REMARKS 

Volume 

Total 
Nitro- 
gen 

Dex- 
trose 

Total 
Solids 

Dex- 
trose 

cc. 

grams 

grams 

per  cent 

per  cent 

November 

29 

100 

0.56 

2.30 

No  water  intake. 

30 

90 

1.31 

2.57 

No  water  intake. 

December 

1 

110 

1.26 

1.73 

Water  intake  =  100  cc. 

2 

5 

0.018 

0.015 

Subcutaneous  injection  of  1 .5 

grams  tartaric  acid  neu- 

tralized with  Na2Co3,  dis- 

solved in  30  cc.  water. 

3 

30 

0.066 

0.068 

Water  intake  =  60  cc. 

4 

20 

trace 

16.61 

0.18 

Blood  drawn  5  hours  after 

last  phlorhizin  injection. 

The  liver  contained  0.35 

gram  glycogen. 

24  Mechanism  of  Phlorhizin  Diabetes 


TABLE  5. 

Experiment  3,  Rabbit  I.  Female  rabbit  of  2300  grams  received  daily  subcu- 
taneous injection  of  0.25  gram  phlorhizin. 


DATE 
1911 

URINE 

BLOOD 

REMARKS 

Volume 

Total 
Nitro- 
gen 

Dex- 
trose 

1  otal 
Solids 

.Dex- 
trose 

cc. 

grams 

grams 

per  cent 

per  cent 

December 

5 

100 

2.88 

4.03 

Water  intake  =  40  cc. 

6 

200 

1 .73 

3.45 

Water  intake  =  125  cc. 

7 

90 

1.84 

1.40 

Water  intake  =  50  cc. 

Q 
O 

10 

0.075 

0.076 

Subcutaneous  injection  of  2.0 

grams  tartaric  acid,  neu- 

tralized with  Na2Coz,  dis- 

solved in  30  cc.  water. 

9 

0 

18  0 

0.21 

Water   intake  =  150  cc. 

Blood   drawn  2.5  hours 

after  last  phlorhizin  in- 

jection.   The  liver  con- 

tained 0.41  gram  glyco- 

gen. 

TABLE  6. 

Experiment  4,  Rabbit  J.    Female  rabbit  of  2800  grams  received  daily  subcu- 
taneous injection  of  0.25  gram  phlorhizin. 


DATE 

1911 

URINE 

BLOOD 

REMARKS 

Volume 

Total 
Nitro- 
gen 

Dex- 
trose 

Total 
Solids 

Dex- 
trose 

cc. 

grams 

grams 

per  cent 

per  cent 

December 

5 

250 

1.45 

2.54 

Water  intake  =  170  cc. 

6 

175 

2.70 

4.46 

Water  intake  =   80  cc. 

7 

125 

2.38 

2.75 

Water  intake  =   90  cc. 

8 

10 

0.075* 

0.076 

Subcutaneous  injection  of  2.0 

grams  tartaric  acid,  neu- 

tralized with  Na2Coz,  dis- 

solved in  30  cc.  water. 

9 

0 

18.70 

0.15 

Water   intake  =  240  cc. 

Blood  drawn  2.5  hours 

after  last  phlorhizin  in- 

jection.   The  liver  con- 

tained 0.60  gram  glyco- 

gen. 

Frank  P.  Underbill 


25 


dogs  the  blood  sugar  content  increased  above  the  normal  after 
practical  extirpation  of  the  kidneys.  It  is  difficult  to  comprehend 
how  extensive  kidney  secretory  activity  could  be  alleged  in  the 
rabbit  experiments  carried  through  in  the  manner  described.  It- 
is  evident  therefore  that  under  the  experimental  conditions  here 
outlined  little  support  can  be  derived  in  favor  of  the  view  advanced 
by  Pavy.  On  the  other  hand,  in  view  of  the  new  facts  furnished 
by  the  present  investigation  the  conception  of  v.  Mering  with 
respect  to  the  nature  of  the  mechanism  of  phlorhizin  diabetes  has 
been  supplemented  and  extended. 

table  7. 


Experiment  5,  Rabbit  K.    Female  rabbit  of  2600  grams  received  daily  subcu- 
taneous injection  phlorhizin. 


URINE 

BLOOD 

DATE 

1911 

Volume 

Total 
Nitro- 
gen 

Dex- 
trose 

Total 
Solids 

Dex- 
trose 

REMARKS 

cc. 

grams 

grams 

per  cent 

per  cent 

December 

11 

85 

1.13 

2.40 

Water  intake  =  25  cc.  In- 
jected 0.25  gram  phlor- 
hizin. 

12 

100 

1.35 

1.58 

Water  intake  =  70  cc.  In- 
jected 0.25  gm.  phlorhi- 
zin. 

13 

10 

0.015 

0.008 

Subcutaneous  injection  of  2.0 
grams  tartaric  acid,  neu- 
tralized with  Na2Co3,  dis- 
solved in  15  cc.  water,  in- 
jected 1.0  GRAM  PHLOR- 
HIZIN. Water  intake  = 
195  cc. 

14 

0 

18  65 

0  20 

Blood  drawn  3.5  hours  after 
injection  of  1.0  gram 
phlorhizin.  The  liver  was 
glycogen  free. 

26  Mechanism  of  Phlorhizin  Diabetes 

TABLE  8. 


Experiment  6,  Rabbit  L.    Female  rabbit  of  2800  grams  received  daily  subcu- 
taneous injection  of  phlorhizin. 


DATE 
1911 

URINE 

BLOOD 

REMARKS 

Total 
Volume  Nitro- 

i  gen 

Dex- 
trose 

Total 
Solids 

Dex- 
trose 

cc. 

grams 

grams 

per  cent 

per  cent 

December 

11 

120 

1.71 

3.30 

Water  intake  =  50  cc. 

12 

120 

2.21 

2.01 

Water  intake  =  50  cc. 

13 

2 

0 

0 

Subcutaneous  injection  of  S.O 

grams  tartaric  acid,  neu- 

tralized with  NaiCoz,  dis- 

solved in  20  cc.  water,  in- 

jected 1.0  GRAM  PHLOR- 

HIZIN.    Water  intake  = 

150  cc. 

14 

0 

16  70 

0  25 

Blood  drawn  4  hours  aft- 

er injection  of  1.0  gram 

phlorhizin.  The  liver  was 

glycogen  free. 

SUMMARY. 

The  mechanism  of  phlorhizin  diabetes  has  been  subjected  to 
investigation  after  the  removal  of  the  renal  secretory  function  by 

(a)  ligation  of  the  renal  structures  in  the  dog  and  (b)  abolition 
of  kidney  secretion  through  subcutaneous  administration  of  sodium 
tartrate  to  rabbits. 

In  both  conditions  a  significant  hyperglycaemia  may  be  in  evidence. 
With  dogs  this  is  accompanied  by  a  decrease  in  the  proportion  of 
solids  in  the  blood,  that  is,  the  water  content  is  increased. 

The  data  presented  lead  to  the  suggestion  that  phlorhizin  may 
possess  a  two-fold  action  (a)  an  influence  is  exerted  upon  the  kidney 
whereby  this  organ  becomes  more  permeable  for  blood  sugar  and 

(b)  an  action  upon  other  structures  resulting  in  the  production 
of  sugar  in  quantities  sufficient  to  cause  hyperglycaemia  if  the 
kidney  function  is  removed. 


Reprinted  from  The  Journal  of  Biological  Chemistry,  Vol.  XIII,  No.  I,  1912. 


THE  BEHAVIOR  OF  FAT-SOLUBLE  DYES  AND  STAINED 
FAT  IN  THE  ANIMAL  ORGANISM.1 

By  LAFAYETTE  B.  MENDEL  and  AMY  L.  DANIELS. 

(From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  August  26,  1912.) 


Introduction   71 

Deposition  of  fat-soluble  dyes  in  animal  tissues   72 

Availability  of  stained  fat  in  metabolism   81 

The  fate  of  fat-soluble  stains  in  the  organism   84 

Fat-transport  in  starvation  and  pathological  conditions   90 

Fat-transport  to  the  embryo   91 

Fat-transport  into  milk   92 

Summary   94 

Bibliography  ,   95 


INTRODUCTION. 

Since  Daddi2  discovered  that  Sudan  III,  when  fed  incorporated 
with  fat,  is  absorbed  and  laid  down  in  the  adipose  tissue  of  animals, 
various  experimenters  have  used  the  dye  as  a  means  of  studying 
problems  connected  with  fat  metabolism.  The  possibilities  of 
this  method  have  not  been  exhausted,  and  the  present  investigation 
was  aimed  to  extend  the  application  of  fat-soluble  dyes  to  the  solu- 
tion of  some  of  the  unanswered  questions. 

The  dyes  used  were :  Sudan  III  (Kahlbaum) ;  Biebrich  Scarlet 
(Aniline  Red,  R.  Medicinal.  Merck) ;  Indophenol  (H.  A.  Metz  and 
Company);  Oil  Soluble  Green  (H.  A.  Metz  and  Company);  Oil 

1  A  preliminary  report  of  some  of  the  data  recorded  here  was  presented 
to  the  Society  for  Experimental  Medicine  and  Biology  (cf.  Proceedings, 
viii,  p.  126,  1911).  The  essential  facts  in  this  paper  are  taken  from  the 
dissertation  presented  by  Amy  L.  Daniels  for  the  degree  of  Doctor  of  Phil- 
osophy, Yale  University,  1912. 

2  Arch.  Hal.  de  biol.,  xxvi,  142,  1896. 

7i 


72 


Behavior  of  Fat-Soluble  Dyes 


Orange  (National  Aniline  and  Chemical  Company);  Blue  Base 
(Hudson  River  Aniline  Color  Works);  Dandelion  Brand  Butter 
Color  (Wells,  Richardson  and  Company);  and  Annatto.  These 
are  water-insoluble  compounds  which  are  soluble  in  fat,  fatty  acids, 
alcohol,  ether,  chloroform,  benzene  and  bile,  as  well  as  in  solutions 
of  the  isolated  bile  salts.  They  were  introduced,  dissolved  in  fat 
or  in  lecithin  emulsions  of  oil,3  either  by  feeding  or  by  intravenous, 
subcutaneous  or  intraperitoneal  injections.  The  dyes  deposited 
in  the  fatty  tissue  and  secreted  milk  of  the  experimental  animals 
were  easily  detected  by  the  color;  those  in  the  glandular,  muscular, 
and  nervous  tissue,  and  in  the  fluids  of  the  body — the  blood,  lymph 
and  bile — were  less  easily  determined.  In  all  cases  2-gram  por- 
tions of  the  tissue  to  be  examined  were  minced,  dried  with  anhy- 
drous sodium  sulphate  and  extracted  with  ether.  The  ether  ex- 
tracts were  filtered,  allowed  to  evaporate  in  white  porcelain 
dishes,  and  the  colors  of  the  residues  were  noted.  The  blood  and 
lymph  were  also  dried  with  anhydrous  sodium  sulphate;  the  bile 
was  similarly  extracted  with  ether  after  being  dried  down  with 
calcium  oxide  to  form  ether-insoluble  compounds  of  the  bile  pig- 
ments. 

DEPOSITION   OF   FAT-SOLUBLE   DYES  IN  ANIMAL  TISSUES. 

With  the  exception  of  the  meal  worm,  Tenebrio  molitor,4  the 
infusoria  (Staniewicz)  and  possibly  the  cow5  the  adipose  tissue 
has  been  found  to  be  stained  in  those  animals  into  which  fat, 
stained  with  Sudan  III,  has  been  introduced.  The  animals  investi- 
gated, the  methods  of  introducing  the  stain  and  the  results  ob- 
tained, are  summarized  in  the  table  on  pages  74  and  75. 

Although  the  time  required  to  stain  the  adipose  tissue  of  animals 
of  different  species  has  been  noted  only  incidentally,  it  would  seem 
from  the  results  reported  that  it  varies  considerably.  Riddle  has 
observed  that  rabbits  and  turtles  absorb  Sudan  III  less  rapidly  than 
the  fowl,  in  which  the  fatty  tissue  is  colored  after  one  or  two  days' 
feeding.    The  red  stain  appeared  in  the  yolk  of  the  eggs  of  hens, 

3  This  emulsion,  supplied  by  Fairchild  Bros,  and  Foster,  consisted  of 
5  per  cent  lecithin,  45  per  cent  peanut  oil  and  50  per  cent  water. 

4  Biedermann:  Arch.  f.  d.  ges.  Physiol.,  lxxii,  p.  105,  1898. 

6  S.  H.  and  S.  P.  Gage:  Science,  xxviii,  p.  494,  1908;  Anatomical  Record, 
hi,  1909. 


Lafayette  B.  Mendel  and  Amy  L.  Daniels  73 


and  in  the  milk  of  rats  after  one  feeding,  whereas  the  cow  observed 
by  S.  P.  and  S.  H.  Gage6  gave  no  evidence  of  Sudan  absorption 
after  four  days  of  Sudan  feeding. 

Experimental.  Feeding  experiments  with  Sudan  III  were 
carried  out  with  rats,  cats,  guinea  pigs,  pigeons,  hens,  frogs,  a  cow 
and  a  goat.  The  results  were  comparable  with  those  of  the  earlier 
investigators.  After  a  single  feeding  of  deeply  stained  food,  colored 
fat  was  found  in  the  milk  of  cats  and  rats,  and  in  the  egg  of  the  hen. 
Pigeons,  after  five  days,  showed  a  distinct  pink  coloration  of  the 
subcutaneous  tissue  through  the  skin;  at  autopsy,  the  fatty  tissue 
was  found  to  be  distinctly  stained.  Three  guinea  pigs,  to  which 
were  given  two  gelatin  capsules,  each  containing  80  mgms.  of 
Sudan  III,  every  second  day  for  four  weeks,  gave  no  evidence  of 
stained  tissue;  two  guinea  pigs,  given  2  cc.  of  stained  oil  every 
second  day  for  three  weeks,  contained  faintly  pink  adipose  tissue. 
Frogs  were  fed  for  three  weeks  during  the  hibernating  period  with 
meat  liberally  mixed  with  stained  oil;  throughout  the  experiment 
they  were  kept  in  a  room  at  20°C.  In  no  case  did  the  fatty  tissue 
of  these  become  stained.  The  cow  secreted  no  stained  milk  even 
after  seven  successive'  feedings  of  7.5  grams  of  Sudan  dissolved 
in  oil;  whereas  the  milk  of  the  goat  was  faintly,  but  distinctly,  pink 
after  one  feeding  of  Sudan-stained  food. 

It  will  be  observed  that,  in  general,  those  animals  (rats,  cats  and 
fowls)  which  absorb  fat  readily,  give  evidence  of  Sudan-stained 
fat  in  less  time  than  those,  like  the  guinea  pig  and  cow,  in  which 
fat  forms  a  smaller  factor  in  the  diet. 

Biebrich  Scarlet,  which  resembles  Sudan  III  in  its  solubilities, 
and  is  not  affected  by  dilute  solutions  of  acids  and  alkalies,  was 
fed  to  pigeons,  rats  and  cats,  with  results  comparable  with  those 
obtained  with  Sudan  III.  The  subcutaneous  tissue  was  colored 
pink. 

Feeding  experiments  with  indophenol-blue  were  unsuccessful. 
This  dye,  unaffected  by  dilute  alkalies,  changes  to  pale  yellowish 
green  when  treated  with  dilute  hydrochloric  acid.  This  color 
change  in  the  stomach  made  it  impossible  to  detect  the  dye  ab- 
sorbed. In  rabbits  and  pigeons  after  subcutaneous  injections  of 
oil  emulsions  colored  with  the  blue  dye  no  fatty  tissue  was  found 

6  Anatomical  Record,  iii,  1909. 


74 


Behavior  of  Fat-Soluble  Dyes 


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76 


Behavior  of  Fat-Soluble  Dyes 


stained.  The  blood  of  rabbits  taken  from  two  to  six  hours  after 
intravenous  injections  of  indophenol-blue  dissolved  in  oil  emulsion 
yielded  pink  residues  on  extraction. 

These  results  point  to  the  reduction  of  the  indophenol-blue  to 
indophenol  by  the  tissues.  The  presence  of  active  reductases  in 
the  various  tissues  of  the  animal  body  has  been  observed  by  Ehr- 
lich,7  Herter,8  Harris9  and  others.  Heffter10  reports  that  the 
liver  is  particularly  rich  in  this  enzyme,  a  fact  which  was  further 
demonstrated  by  us  as  follows: 

Ground  liver  tissue,  to  which  oil  stained  with  indophenol-blue  had 
been  added,  was  allowed  to  autolyze  in  the  presence  of  toluene  at  a  tem- 
perature of  30°C.  After  twenty-four  hours,  the  mixture  had  lost  its  blue 
color  and  had  become  pink;  the  addition  of  hydrogen  peroxide  brought 
back  the  blue  color.  No  change  in  color  took  place  in  a  control  experi- 
ment, carried  out  with  boiled  liver  tissue  under  identical  conditions. 

i 

The  localization  of  fat-soluble  dyes  in  the  tissues. 

Analysis  of  the  various  tissues  of  the  animal  body  shows  that 
the  largest  quantity  of  fat  (ether  extract)  is  found  in  the  subcuta- 
neous tissue,  the  fatty  tissue  of  the  abdominal  cavity  and  the  bone 
marrow;  however,  the  muscular,  glandular  and  nervous  tissues 
contain  estimable  amounts.  It  is  reasonable  to  suppose,  therefore, 
that  animals  containing  Sudan-stained  adipose  tissue  would  like- 
wise have  stained  fat  in  the  other  fat-bearing  tissues,  especially 
since  this  dye  readily  reveals  the  presence  of  fat  in  histological 
sections  of  these  tissues.  The  only  investigators  who  even  suggest 
that  the  fat  of  other  than  the  adipose  tissues  may  not  be  colored 
are  Mann11  and  S.  P.  and  S.  H.  Gage.12  The  basis  for  Mann's 
statement  that  "  animals  fed  on  oil  colored  with  Sudan  III  show 
only  the  adipose  tissue  stained"  is  not  clear.  S.  P.  and  S.  H.  Gage 
failed  to  find  the  stain  in  the  nerve  fibres  of  the  chicks  developed 
from  the  Sudan-stained  eggs,  although  the  adipose  tissues  of  these 
were  distinctly  colored. 

7  Das  Sauerstoffbediirfniss  des  Organismus,  Berlin,  1885. 

8  Amer.  Journ.  of  Physiol.,  xii,  pp.  207,  457,  1904-5. 

9  Bio-chem.  Journ.,  v,  p.  143,  1911. 

10  Medizinisch  naturwissenschaftliches  Archiv,  i,  p.  81,  1907-8. 

11  Physiological  Histology,  1902,  pp.  36-7. 

12  Science,  xxviii,  p.  494,  1908. 


Lafayette  B.  Mendel  and  Amy  L.  Daniels  77 


Bondi  and  Neumann18  found  that  the  bone  marrow  and  livers 
of  rabbits  were  distinctly  blue  after  the  injection  of  an  emulsion 
of  fat,  stained  with  indophenol,  and  that  the  Kupfer  cells  of  the 
•livers  of  rabbits  became  distinctly  pink  after  the  injection  into 
the  circulation  of  an  oil  emulsion  stained  with  Scharlach  Rot.  The 
animals  were  killed  a  few  hours  after  the  injection;  the  adipose 
tissue  had  not  become  stained  in  this  short  time,  and  the  fact  that 
the  liver  cells  contained  the  color  of  the  dye  injected  cannot  be 
taken  as  proof  that  these  cells  store  fat.  The  results  of  subsequent 
experiments  in  this  investigation  pertaining  to  the  mode  of  elimi- 
nation of  fat-soluble  dyes,  to  which  reference  will  be  made  later, 
have  thrown  some  light  upon  this  point,  and  make  it  evident  that 
these  observations  of  Bondi  and .  Neumann  may  be  otherwise 
interpreted. 

Expekimental.  In  order  to  ascertain  whether  stained  fat, 
other  than  that  in  the  distinctly  adipose  tissue,  is  present  in  the 
bodies  of  animals  into  which  fat-soluble  dyes  have  been  introduced, 
2-gram  portions  of  the  tissues  to  be  examined  were  freed,  as 
far  as  possible,  from  extraneous  fat  and  connective  tissue,  finely 
divided,  dried  and  extracted  with  ether  in  accordance  with  the 
method  already  described.  The  dyes  were  administered  dissolved 
in  olive  oil  or  in  lecithin  emulsion  of  peanut  oil.  The  results  are 
summarized  in  the  table  on  pages  78  and  79. 

Discussion.  Negative  results  were  always  obtained  from 
nervous  and  renal  tissues;  from  muscle  when  it  was  freed  from  con- 
nective tissue  or  extraneous  fat  as  in  starvation:  and  in  general 
from  liver  tissue.  Livers  however  from  which  blood  had  not  been 
removed  by  perfusion  or  bleeding  sometimes  showed  traces  of  the 
dye.  In  two  cases  the  livers  from  rats  which  had  been  fed  on  a 
diet  containing  75  per  cent  of  deeply  stained  lard,  yielded  con- 
siderable quantities  of  the  dye.  These  livers  were  distinctly 
pink,  owing  undoubtedly  to  the  storage  of  the  absorbed  fat  in  the 
liver  cells.  Microscopic  examinations  of  frozen  sections,  however, 
failed  to  disclose  the  dyes,  even  when  chemical  isolation  demon- 
strated their  presence.  . 

The  explanation  of  these  results  is  not  clear.  It  may  be  that 
the  form  of  the  fat  in  the  nervous,  muscular  and  glandular  tissues 

13  Zentralbl.  f.  Biochem.  u.  Biophysik,  x,  p.  1453,  1910. 


78 


Behavior  of  Fat-Soluble  Dyes 


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Behavior  of  Fat-Soluble  Dyes 


of  the  body  is  quite  different  from  that  in  the  adipose  tissue — that 
it  is  held  in  some  loose  chemical  combination  which  is  no  longer 
capable  of  taking  up  the  stain.  The  present  methods  of  fat  extrac- 
tion and  staining  may  result  in  a  disintegration  of  this  complex 
molecule.  MacLean  and  Williams14  have  advanced  the  theory 
that  the  fat  removed  by  extraction  from  animal  tissues  does  not 
represent  the  form  in  which  the  fat  exists  in  these  tissues,  and 
that  the  fat  is  made  evident  as  the  result  of  certain  post-mortem 
changes  by  which  the  compound  is  broken  up  and  the  fat  liberated. 
Leathes15  and  Abderhalden  and  Brahm16  have  suggested  that  the 
fat  of  the  active  tissues  differs  from  that  of  the  storage  tissue.  In 
the  present  investigation  it  was  found  that  the  isolated  ether-soluble 
substances  of  the  brain  can  take  up  the  stain.  This  observation, 
together  with  the  fact  that  the  nervous  tissue  of  Sudan-stained 
animals  is  always  free  from  the  dye,  even  when  the  embryonic  fat 
contained  an  abundance,  as  was  demonstrated  by  S.  P.  and  S.  H. 
Gage17  in  chicks  developed  from  the  stained  eggs,  adds  weight  to 
the  theory  outlined  above. 

An  explanation  of  the  fact  that  in  a  large  number  of  the  experi- 
ments the  liver  tissue  was  found  to  be  free  from  the  dye,  is  afforded 
by  the  observation  that  the  fat-soluble  dyes  are  more  soluble  in  bile 
than  in  fat;  and  when  these  dyes  are  introduced  into  the  body  in  solu- 
tion in  the  fat  they  are  eliminated  in  the  bile.  Added  evidence  in 
favor  of  this  explanation  is  found  in  the  fact  that  the  fat  complex 
in  the  liver  is  not  incapable  of  holding  the  dye  in  combination. 
This  is  shown  by  the  following  experiment: 

A  solution  of  Sudan  III  in  bile  was  injected,  under  pressure,  into  the 
common  bile  duct  of  a  rabbit.  After  20  cc.  had  been  forced  in,  the  liver 
was  removed,  perfused  with  physiological  saline  solution,  comminuted, 
washed  in  cold  running  water  for  twenty-four  hours  and  filtered;  the  resi- 
due, which  was  distinctly  pink,  was  washed  until  the  filtrate  gave  no  test 
for  bile  salts  with  Pettenkofer's  reaction;  ether  extracts  of  the  dried  residue 
were  distinctly  pink.  There  could  be  no  doubt  that  the  fat  had  absorbed 
the  stain. 

Conclusions.  Stained  fats,  introduced  into  the  animal  body 
intraperitoneally,  intravenously,  subcutaneously  or  by  absorption 

14  Bio-chem.  Journ.,  iv,  p.  455,  1909. 

16  Problems  in  Animal  Metabolism,  1906,  p.  72. 

16  Zeitschr.  f.  physiol.  chem.,  lxv,  p.  330,  1910. 

17  Science,  xxviii,  p.  494,  1908. 


Lafayette  B.  Mendel  and  Amy  L.  Daniels  81 


from  the  alimentary  tract,  are  laid  down  in  the  adipose  tissue  and 
marrow. 

The  renal  and  nervous  tissues  are  free  from  the  stain,  even  when 
the  fatty  tissue  is  deeply  colored;  muscle  tissue,  when  freed  from 
fat,  as  in  extreme  starvation,  contains  no  stained  fat;  the  dye  is 
found  in  the  liver  only  when  the  blood  contains  an  abundance,  as 
in  starvation,  or  when  the  animal  has  been  fed  food  containing  a 
large  amount  of  stained  fat  a  few  hours  before  the  examination. 

Liver  fat,  in  situ,  is  capable  of  taking  up  the  stain. 

Indophenol-blue  is  reduced  in  the  body;  this  reduction  takes 
place,  in  part,  in  the  liver;  hence  adipose  tissue  is  not  stained  with 
this  dye. 

AVAILABILITY  OF  STAINED  FAT  IN  METABOLISM. 

Riddle18  has  suggested  that  adipose  tissue  stained  with  Sudan 
III  is  less  available  to  the  organism  than  unstained  adipose  tissue. 
Inasmuch  as  the  dye  enters  into  no  chemical  union  with  the  fat, 
but  is  merely  dissolved  therein,19  it  does  not  seem  probable  that 
the  Sudan  III  can  so  change  the  nature  of  the  fat  that  it  cannot 
be  used  as  effectively  by  the  organism  as  unstained  fat.  An  indif- 
ferent material,  like  Sudan  III,  might  be  toxic,  or  might  form  toxic 
combinations  in  the  body,  and  thus  affect  organs  dealing  with  fat 
combustion;  but  that  the  fat  itself  is  rendered  unavailable  scarcely 
seems  tenable.  The  non-toxicity  of  Sudan  III  has  been  shown 
by  feeding  animals  over  long  periods  of  time  without  apparent 
deleterious  results. 

Experimental.  In  order  to  determine  if  Sudan-stained  fat 
is  less  available  to  the  organism,  starvation  experiments  were 
carried  out  with  Sudan-stained  rats  and  pigeons;  comparable  experi- 
ments were  conducted  with  normal  animals. 

1.  Pigeon  B.  Fed  with  pulverized  dog  biscuit,  lard  deeply  stained 
with  Sudan  III,  and  cracked  corn  for  three  weeks  before  the  beginning  of 
the  fasting  period.  Subcutaneous  tissue  became  pink.  Weight  of  pigeon 
at  beginning  of  fast  was  297  grams.  Death  in  ten  days.  It  had  lost  116.5 
grams,  39  per  cent  of  its  initial  weight;  all  visible  fat  had  disappeared. 
No  pink  color  was  to  be  seen.    A  slight  trace  of  Sudan  III  was  found  in 


18  Journ.  of  Exp.  Zobl.,  viii,  p.  163,  1910. 
19Michaelis:  Virchoiv's  Archiv.,  clx,  p.  263,  1901. 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  XIII,  NO.  1. 


82 


Behavior  of  Fat-Soluble  Dyes 


ether  extract  of  the  tail  gland,  bone  marrow  and  liver;  the  muscle,  kidney 
and  brain  contained  no  trace  of  the  dye. 

2.  Pigeon  C.  Preliminary  feeding  same  as  B.  Subcutaneous  tissue 
became  noticeably  pink.  Weight  at  beginning  of  fast  294  grams.  Death 
in  eleven  days.  Loss  of  weight  165  grams,  56  per  cent.  No  visible  fat 
remained;  tissues  showed  no  pink  color;  ether  extract  of  liver  and  bone 
marrow  slightly  pink;  of  muscle,  kidney  and  brain,  colorless. 

3.  Pigeon  D.  Fed  Sudan-stained  food  as  in  B  and  C.  After  sixteen  days 
of  fasting  this  animal  died.  It  had  lost  41  per  cent  of  its  initial  weight. 
All  visible  fat  and  stain  had  disappeared  from  the  body.  The  ether  extract 
of  the  bone  marrow  was  slightly  pink;  that  from  the  liver  and  muscle  showed 
no  pink  color. 

4.  Pigeon  E.  A  normal  well-fed  bird,  was  starved  for  sixteen  days,  dur- 
ing which  it  lost  229  grams  or  54  per  cent  of  its  initial  weight.  All  visible 
fat  had  disappeared  from  the  body. 

5.  Pigeon  F.  A  well-fed  normal  bird  which  died  fifteen  days  after  the 
fasting  period  began.  The  loss  in  weight  was  144  grams  or  45  per  cent  of 
its  initial  weight.    All  visible  fat  had  disappeared. 

It  should  be  noted  that  Pigeons  B  and  C  were  kept  during  November 
in  an  unheated  room  with  the  windows  open.  This  doubtless  explains  their 
earlier  death  as  compared  with  pigeons  D,  E  and  F  which  were  kept  at 
about  20°C.  In  every  case,  however,  the  fatty  tissue  had  entirely  disap- 
peared from  the  body. 

Experiments  with  rats  gave  similar  results. 

6.  Rat  A.  Fed  with  ground  dog  biscuit  mixed  with  lard  deeply  stained 
with  Sudan  III  for  seven  days.  Fasting  period,  three  days.  Loss  in  weight, 
42  grams,  42  per  cent  of  initial  weight.  The  body  was  free  from  all  traces 
of  visible  fat  and  stain. 

7.  Rat  C.  Preliminary  feeding  period,  same  as  A.  Fasting  period  ap- 
proximately sixty  hours.  All  visible  fat  disappeared  from  the  body.  The 
ether  extract  of  the  brain,  liver,  muscle,  kidney  and  subcutaneous  tissue 
left  no  pink  residue. 

8.  Rat  B.  A  normal  well-fed  rat,  which  died  after  a  60  hours'  fast.  The 
loss  in  weight  was  40  grams  or  22  per  cent  of  its  initial  weight.  The  body 
was  free  from  all  visible  fat. 

9.  Control  experiment.  Rat  D.  Was  fed  on  Sudan-stained  food  as  in 
the  previous  experiments,  for  seven  days.  The  subcutaneous  tissue,  omen- 
tum and  fatty  tissue  about  the  kidneys  were  deeply  pink. 

The  result  of  the  control  experiment  affords  evidence  tljat  the 
adipose  tissue  of  the  experimental  animals  was  similarly  stained 
at  the  beginning  of  the  fasting  periods.  The  further  observation 
was  made  that  rats,  and  in  some  cases  rabbits,  stained  as  the  result 
of  feeding  with  deeply  stained,  fat-rich  food,  excreted  urines  which 


Lafayette  B.  Mendel  and  Amy  L.  Daniels  83 


were  distinctly  pink;  such  urines  were  found  to  contain  both  fat 
and  Sudan  III. 

In  two  experiments  Sudan-stained  pigeons  were  fed  with  un- 
stained foods  after  long  fasting  periods — other  pigeons,  fasting  the 
same  length  of  time  and  under  similar  conditions,  had  died.  At 
autopsy,  the  fatty  tissue  of  these  was  found  to  be  unstained. 

10.  Pigeon  G.  Fed  with  Sudan-stained  fat,  described  in  protocols  1-3, 
starved  thirteen  days;  loss  of  weight,  104  grams  or  29  per  cent.  It  was 
re-fed  and  examined  some  months  later.  All  trace  of  Sudan  had  disap- 
peared; the  ether  extract  of  the  tissues  left  no  pink  residue. 

11.  Pigeon  A.  Previously  fed  with  Sudan-stained  food;  starved  eleven 
days;  loss  of  weight,  104  grams  or  32  per  cent.  It  was  re-fed  until  it  had 
gained  16  grams.  Upon  examination,  no  stained  tissue  was  found.  Ether 
extract  of  subcutaneous  fat,  tail  gland  and  omentum  showed  no  pink  color. 

Discussion.  The  results  of  these  trials  are  not  in  agreement 
with  those  reported  by  Riddle.  Sudan-stained  pigeons  and  rats 
died  in  no  less  time  than  the  unstained  control  animals.  In  both 
cases  the  visible  fat  had  entirely  disappeared,  and,  in  the  stained 
animals,  the  dye  as  well.  Those  animals  which  were  fed  after 
long  fasting  periods  until  there  was  a  marked  increase  in  body 
weight,  contained  no  trace  of  the  former  Sudan-stained  fatty  tissue. 
One  must  conclude  from  these  results  that  stained  adipose  tissue 
is  no  less  available  to  the  organism  than  the  non-Sudan-stained 
fat  and  that  it  is  used  quite  as  readily  and  completely. 

The  disparitybetween  our  results  and  those  of  Riddle  is  difficult 
to  explain.  His  observations  that  chicks  fed  on  stained  food 
developed  more  slowly  than  normal  chicks  and  that  hens  ceased 
to  lay  after  considerable  quantities  of  the  dye  had  been  ingested 
may  have  resulted  from  other  causes  than  the  ingestion  of  the 
dye.  It  is  conceivable  that  the  apparent  failure  of  starving  stained 
animals  in  his  experiments  to  utilize  their  fatty  tissues  as  do  normal 
animals  was  the  result  of  impurities  in  the  dye  fed.  Mann20  has 
observed  that  Scharlach  Rot  given  to  half  grown  kittens  in  large 
doses  causes  vomiting.  We  gave  large  doses  of  Sudan  III,  put 
up  by  an  American  manufacturer,  to  two  cats.  These  died  within 
a  comparatively  short  time  apparently  from  the  effect  of  some  im- 
purity in  the  dye.  Other  cats,  given  equally  large  doses  of  the 
Kahlbaum  dye,  experienced  no  ill  effects.  Riddle's  deductions  from 

20  Personal  communication. 


84 


Behavior  of  Fat-Soluble  Dyes 


his  second  series  of  fasting  experiments  that  stained  animals  under- 
went a  greater  percentage  loss  of  weight  during  starvation  than  do 
unstained  are  unconvincing  by  reason  of  the  fact  that  an  important 
part  of  his  weighing  records  was  lost. 

THE  FATE  OF  FAT-SOLUBLE  DYES  IN  THE  ORGANISM. 

The  observations  cited  above  have  shown  that  Sudan  III,  depos- 
ited in  the  tissues  as  the  result  of  adding  the  dye  to  the  food,  dis- 
appears completely  during  starvation.  Experiments  upon  cats 
and  rats  gave  no  reasons  for  thinking  that  this  disappearance  is 
due  to  elimination  of  the  dye  by  the  kidneys.  The  fact  that  the 
excreta  of  starving  Sudan-stained  pigeons  contained  the  dye  and 
the  observation  that  the  dye  was  present  in  the  gall  bladders  of 
Sudan-stained  animals  subjected  to  starvation  or  poisoning  with 
phosphorus  or  phlorhizin  turned  our  attention  first  to  the  elimina- 
tion of  fat-soluble  dyes  by  way  of  the  bile.  It  is  well-known  that 
the  bile  is  the  normal  path  of  elimination  of  many  substances. 
From  the  work  of  Abel  and  Rountree21  on  phenoltetrachlor- 
phthalein  the  assumption  seems  justifiable  that  substances  which 
leave  the  body  exclusively  by  way  of  the  bile  must  be  insoluble 
in  water  and  soluble  in  bile  or  substances  contained  therein. 

Two  preliminary  experiments  were  made  upon  cats,  previously 
fed  with  Sudan  III  and  starved  for  four  days  preceding  the  experi- 
ment. The  bile,  collected  as  it  was  secreted  by  the  liver,  and  the 
blood  yielded  pink  ether  extracts,  while  those  obtained  from  the 
liver  tissue,  washed  free  of  blood  and  bile,  were  colorless.  These 
results  pointed  to  a  transport  of  Sudan-stained  fat  to  the  liver  with 
subsequent  storage  of  the  fat  in  the  liver  and  elimination  of  Sudan 
III  in  the  bile. 

The  elimination  of  fat-soluble  dyes  under  normal  conditions. 

The  dyes,  dissolved  in  lecithin-oil-emulsion,  were  introduced 
into  the  circulations  of  cats,  dogs  and  rabbits  by  injections  into 
the  femoral  veins.  In  each  case  the  urine  contained  in  the  bladders, 
as  well  as  the  liver  tissue  and  bile,  was  examined  for  the  injected 
stain.    The  approximate  time  of  the  appearance  of  the  dye  in  the 

21  Journ.  of  Pharmacol,  and  Exp.  Therapeutics,  i,  p.  231,  1909. 


Lafayette  B.  Mendel  and  Amy  L.  Daniels  85 

bile  after  its  introduction  into  the  blood  stream  was  incidentally 
noted. 

The  results  of  the  experiments  are  summarized  in  the  following 
table : 


The  excretion  of  fat-soluble  dyes  introduced  dissolved  in  fat. 


ANIMAL 

DYE 

COLOR  OP 
DYE 

RESIDUE 
FROM  ETHER 
EXTRACT  OF 
BILE 

TIME  OF  AP- 
PEARANCE 
OF  DYE  IN 
BILE 

DYE  IN 
URINE 

DYE  IN 
LIVER 

TTtitllliCS 

Cat  II,  8 

Indophenol 

Blue 

Blue 

30f 

None 

Dog  II,  14. . .  . 

Sudan 

Red 

Pink 

30 

None 

Present 

Dog  II,  16. . . . 

Sudan  III 

Red 

Pink 

20 

None 

None 

Cat  II,  21. . .  . 

Indophenol 

Blue 

Til 

Blue 

60t 

None 

Present* 

Dog  11,21.... 

Sudan  III 

Red 

Pink 

Cat  II,  22. . .  . 

Oil  Green 

Green 

Green 

30 

None 

Nonef 

Cat  II,  23.... 

Oil  Green 

Green 

Green 

90 

None 

None 

Rabbit  II,  28. 

Biebrich  Scar- 

let 

Red 

Pink 

60 

None 

Present 

Dog  III,  21  .  . 

Sudan  III 

Red 

Pink 

60 

None 

None 

Cat  III,  24. . . 

Sudan  III 

Red 

Pink 

75 

None 

None 

Cat  IV,  21.... 

Butter  Color 

Yellow 

Pink 

55 

None 

None 

Cat  V,  2 

Oil  Orange 

Orange 

Yellow- 

red 

90 

None 

None 

Cat  V,  6 

Blue  Base 

Blue 

Pink 

50 

None 

Present^ 

Cat  V,  15 

Butter  Color 

Yellow 

Pink 

None 

*  The  animal  died  fifteen  minutes  after  the  second  injection  of  dye. 
t  The  animal  died  one-half  hour  after  the  injection. 

t  The  addition  of  dilute  hydrochloric  acid  to  the  liver  tissue  resulted  in  a  blue  color.  The 
animal  died  four  hours  after  the  injection. 


In  a  number  of  the  experiments  the  residues  from  the  ether 
extracts  of  the  excreted  bile  were  examined  for  fat.  The  ethereal 
filtrates  were  allowed  to  evaporate  from  watch  glasses;  the  residues 
were  heated  gently;  no  melting  of  the  material  took  place  and  no 
grease  spot  was  formed  on  soft  tissue  paper  by  this  residue.  The 
dyes  were  excreted  dissolved  in  bile  and  not  in  combination  with 
fat. 

In  some  cases,  the  color  of  the  residues  from  the  ether  extracts 
of  the  bile  was  not  precisely  like  that  of  the  dyes  injected.  This 
change  in  color  is  the  result  of  the  passage  of  the  dyes  through  the 
body  where  they  are  brought  in  contact  with  hydroxylions.  The 
action  of  dilute  alkalies  on  the  dyes  outside  the  body  causes  a  sim- 
ilar change. 


86 


Behavior  of  Fat-Soluble  Dyes 


The  residues  from  the  ether  extracts  of  the  liver  tissue  were  not 
always  colored;  when  the  animals  were  killed  some  time  after  the 
injection  of  the  stain,  or  when  only  a  small  amount  had  been  intro- 
duced, the  liver  was  found  to  be  free  from  the  dye.  The  urines 
were  consistently  free  from  stain. 

It  is  obvious  from  these  results  that  fat-soluble  dyes,  when  intro- 
duced into  the  circulation  in  solution  in  fat,  become  separated  from 
the  fat  and  are  eliminated  in  the  bile. 

Absorption  of  fat-soluble  dyes  into  the  portal  circulation. 

The  next  experiments  were  designed  to  determine  the  roles 
played  by  the  bile  and  by  the  fat  in  the  absorption  of  fat-soluble 
dyes  from  the  intestine. 

22.  Dog:  25  kgm.  Fed  at  7.20  a.m.  Two  hours  later  the  animal  was 
anaesthetized,  and  a  cannula  was  inserted  in  the  thoracic  duct.  Twenty 
cubic  centimeters  of  Sudan-stained  oil  were  injected  into  the  duodenum 
at  11.15  a.m.,  followed  by  1  gram  of  desiccated  ox  bile  in  solution.  At  12.15 
the  lymph  was  intensely  pink.  At  2.00  p.m.,  a  cannula  was  inserted  in  the 
bile  duct.  The  animal  was  killed  by  bleeding  at  5.30  p.m.  Two-gram  por- 
tions of  the  liver  tissue,  10  cc.  samples  of  the  blood,  50  cc.  of  the  lymph  and 
from  2  to  4  cc.  of  the  bile  were  examined.  The  ether  residues  from  the  dried 
lymph  and  bile  were  distinctly  pink,  while  those  from  the  blood  and  liver 
showed  no  trace  of  the  dye. 

23.  Cat,  full-grown.  Fed  at  8.15  a.m.,  was  anaesthetized  at  9.30  a.m. 
A  cannula  was  inserted  in  the  thoracic  duct  at  10.45  a.m.;  after  10  cc.  of 
Sudan-stained  emulsified  oil  had  been  injected  into  the  duodenum,  a  cannula 
was  placed  in  the  common  bile  duct  and  the  bile  collected  therefrom.  The 
lymph  flowed  freely,  and  at  2.30  p.m.  it  was  distinctly  pink  in  color.  The 
ether  residues  from  the  lymph  and  bile  were  distinctly  pink.  The  blood 
(10  cc.)  taken  at  5.00  p.m.  yielded  no  pink  residue.  The  liver  was  also  free 
from  the  dye. 

These  experiments  show  clearly  that  although  Sudan  III,  intro- 
duced with  fat  into  the  intestine,  is  absorbed  by  the  lacteals  and 
appears  in  the  thoracic  lymph,  it  is  still  absorbed  and  eliminated 
in  the  bile,  under  conditions  which  preclude  the  entrance  of  the 
lymph  into  the  blood.  In  the  latter  case,  neither  the  blood  of  the 
general  circulation  nor  the  liver  tissue  is  stained.  This  behavior 
is  explained  in  part  by  the  observation  that  the  dyes  studied  are 
more  soluble  in  bile  than  in  fat  and  by  the  results  of  the  following 
experiments  which  show  that  the  dyes  may  be  absorbed  from  the 
intestine  into  the  portal  circulation  in  solution  in  bile. 


Lafayette  B.  Mendel  and  Amy  L.  Daniels  87 

Pasting  animals  were  anaesthetized,  a  cannula  inserted  into  the 
bile  duct  and  bile  solutions  of  the  various  dyes  used  in  the  earlier 
experiments  were  injected  into  the  small  intestine.  The  results 
are  summarized  below. 


The  excretion  of  fat-soluble  dyes  absorbed  from  alimentary  tract,  dissolved 

in  bile. 


ANIMAL 

DYE 

COLOR  OF 
DYE 

RESIDUE 
FROM  ETHER 
EXTRACT  OF 
BILE 

DYE  IN 
LIVER 

DYE  IN 
BLOOD 

DYE  IN 
URINE 

Cat  II,  8  

Sudan  III 

Red 

Pink 

None 

None 

None 

Cat  II,  8  

Blue  Base 

Blue 

Blue 

None 

None 

Cat  III,  10 

Indophenol 

Blue 

Blue 

None 

None 

None 

Cat  III,  10  

Oil  Green 

Green 

Brown  pink 

None 

None 

None 

Rabbit  III,  20. . . 

Biebrich-Scar- 

let 

Red 

Pink 

None 

None 

Cat  V,  29  

Oil  Yellow 

Orange 

Yellow 

None 

pink 

The  presence  of  the  dye  in  the  bile  in  these  experiments  and  its 
absence  from  the  blood  of  the  general  circulation  show  clearly  that 
it  is  absorbed  with  the  bile  by  the  portal  circulation  and  eliminated 
with  the  bile  by  the  liver.  That  none  of  the  dye  entered  into  the 
general  circulation  is  evidenced  by  the  fact  that  the  blood  of  the 
animals  examined — five  out  of  six— gave  no  indication  of  even 
traces  of  the  dye  when  tested  by  a  method  capable  of  detecting 
0.00001  gram  of  Sudan  III  in  10  cc. 

The  two  following  experiments  show  that  when  bile  is  not  present 
with  the  dye  in  the  intestine,  no  absorption  of  the  dye  in  the  portal 
circulation  occurs. 

Stained  fat  was  introduced  into  a  loop  of  the  upper  intestine 
after  this  had  been  washed  out  with  physiological  saline  solution 
to  remove  all  traces  of  the  adherent  bile.  The  bile  excreted  under 
these  conditions  was  free  from  the  stain,  although  in  one  case  (cf. 
protocol  24)  the  thoracic  lymph  showed  that  a  slight  amount  of 
fat  absorption  had  taken  place;  in  the  other  experiment  (cf.  proto- 
col 25)  both  bile  and  lymph  contained  no  dye  until  after  the  intro- 
duction of  a  bile  solution  into  the  intestinal  loop,  when  the  excreted 
bile  was  found  to  contain  Sudan  III,  although  the  lymph  was  still 
colorless. 


88 


Behavior  of  Fat-Soluble  Dyes 


24.  Dog:  20  kgm.  Narcotized  with  morphine  and  ether.  A  temporary 
lymph  cannula  was  inserted  at  10.00  a.m.,  and  a  bile  cannula  at  10.45  a.m. 
A  12-inch  loop  of  the  intestine  was  tied  off  just  below  the  pylorus  and 
washed  out  with  physiological  saline  solution,  at  body  temperature,  until 
the  washings  were  clear.  Sudan-stained  oil,  together  with  a  solution  of 
0.1  per  cent  HC1,  introduced  to  increase  the  pancreatic  secretion,  were 
injected  into  the  intestinal  loop  at  11.00  a.m.  Bile,  collected  at  12.00  m., 
2.00  p.m.  and  3.30  p.m.,  when  dried  and  extracted,  left  no  pink  residues. 
Seventy  cc.  of  lymph,  collected  between  2.00  and  3.30  p.m.,  contained  a 
small  amount  of  Sudan  III;  the  ether  extract  of  dried  blood  was  unstained. 

25.  Dog:  8  kgm.  Anaesthetized  with  morphine  and  ether  at  9.45  a.m. 
The  insertion  of  the  temporary  lymph  cannula  immediately  preceded  that 
of  the  bile  cannula.  The  intestine  was  ligated  just  below  the  pylorus 
and  14  inches  below  it.  This  loop  was  washed  out  with  physiological 
saline  solution  until  the  washings  were  clear.  Approximately  10  cc.  of 
Sudan-stained  emulsified  oil,  together  with  10  cc.  of  0.1  per  cent  HC1  were 
introduced  into  this  loop.  The  bile  collected  at  3.30  p.m.  left  no  pink  resi- 
due; the  lymph  also  was  free  from  dye.  At  3.30  p.m.,  10  cc.  of  a  solution 
of  desiccated  ox  bile  were  injected  into  the  intestinal  loop.  The  bile  col- 
lected at  7.30  p.m.,  3.5  cc,  showed  the  presence  of  the  dye,  while  the  lymph 
taken  at  this  time,  25  cc,  left  no  pink  color  when  extracted. 

The  elimination  of  the  dye  in  the  bile  during  fat  absorption,  under 
conditions  where  the  stained  fat  was  prevented  from  entering  the 
general  circulation,  was  undoubtedly  due  to  the  migration  of  the 
dye  from  the  fat  to  the  bile  in  the  intestine  and  its  subsequent 
absorption.  There  is  no  reason  to  believe  that  the  dye  in  the 
excreted  bile  was  the  result  of  absorption  of  stained  fat  into  the 
portal  circulation.  Had  such  been  the  case,  the  dye  would  have 
been  present  in  the  excreted  bile  in  experiment  24,  as  well  as  in  the 
lymph. 

The  time  required  for  the  absorption  and  deposition  of  fat,  studied 
by  means  of  fat-soluble  dyes. 

The  fact  that  fat-soluble  dyes  are  eliminated  in  the  bile  explains 
some  hitherto  inexplicable  phenomena  observed  in  the  work  with 
Sudan  III.  Earlier  in  this  investigation  an  attempt  was  made  to 
determine  the  length  of  time  required  to  lay  down  the  fat  absorbed 
from  the  alimentary  tract.  Stained  fat  was  fed  to  rabbits  and 
cats;  and  samples  of  blood,  taken  from  the  ear  veins  of  the  rabbits 
and  the  jugular  veins  of  the  cats,  were  examined  for  the  circulating 
dye.    The  stain  was  still  found  to  be  present  in  the  blood  of  rabbits 


Lafayette  B.  Mendel  and  Amy  L.  Daniels  89 


one  week  after  the  last  stained  feeding;  and  the  blood  of  the  cats; 
tested  from  four  to  five  weeks  after  the  last  Sudan-feeding,  left 
distinctly  pink  residues.  Oil  emulsions  stained  with  Sudan  III  and 
Biebrich  Scarlet  gave  similar  results  when  injected  into  the  cir- 
culation of  rabbits.  The  blood  of  these  was  found  to  contain  the 
dye  three  weeks  after  the  last  injection. 

These  observations  find  their  explanation  in  the  fact  that  dye, 
absorbed  from  the  intestine  into  the  lymph  with  the  fat  and  into 
.  the  portal  blood  with  the  bile,  again  enters  into  the  intestine  with 
the  bile.  Thus  a  closed  circulation  of  the  dye  is  established  and 
it  is  possible  that  the  blood  of  a  once  Sudan-stained  animal  may 
become  quite  free  from  the  stained  fat  only  after  long  periods  under 
normal  conditions  of  feeding.  Animals  examined  months  after 
the  Sudan-stained  feeding  had  ceased  contained  deeply  stained 
fatty  tissue. 

The  time  required  for  the  elimination  of  circulating  fat-soluble  dyes. 

An  attempt  was  made  to  ascertain  (in  cats  and  dogs)  the  length 
of  time  necessary  for  the  separation  of  the  dye  from  the  circulating 
fat  and  its  elimination  through  the  bile.  Emulsions  of  stained 
fat,  in  amounts  varying  from  1  to  10  cc,  wei*3  injected  directly 
into  the  circulation;  cannulae  were  placed  in  the  common  bile 
ducts  and  samples  of  blood  and  bile  were  taken  every  two  or  three 
hours.  In  order  to  facilitate  the  flow  of  bile,  solutions  of  desiccated 
ox  bile  were  injected  into  the  upper  intestine.  The  bile,  blood, 
and  liver  tissue  after  it  had  been  washed  free  from  blood,  so  far  as 
possible,  were  examined  for  the  stain. 

Nine  and  one-half  hours  was  the  longest  period  during  which 
observations  were  made  in  any  experiment;  and  although  in  that 
instance  only  1  cc.  of  the  stained  emulsion  was  injected,  both  blood 
and  bile,  collected  at  the  end  of  this  time,  showed  that  a  consider- 
able quantity  of  stained  fat  was  still  in  circulation.  In  those  cases 
in  which  the  experiments  continued  over  a  comparatively  long 
time,  or  when  a  small  amount  of  the  stained  fat  had  been  intro- 
duced, the  liver  was  free  from  the  stain.  The  liver  evidently  does 
not  store  up  stained  fat;  the  dye  becomes  separated  from  the  fat 
as  the  stained  fat  comes  in  contact  with  the  bile  in  the  liver  cells. 

Discussion.  Fat-soluble  dyes  introduced  into  the  body  in 
solution  in  fat  are  secreted  in  the  bile.    These  dyes  may  enter  the 


90  Behavior  of  Fat-Soluble  Dyes 

body  from  the  alimentary  tract  in  two  ways:  (1)  in  the  lymph,  in 
solution  in  fat;  (2)  through  the  portal  circulation,  dissolved  in 
reabsorbed  bile.  When  the  dyes  are  absorbed  dissolved  in  bile, 
they  apparently  do  not  pass  beyond  the  liver,  but  are  speedily 
reexcreted  into  the  gut,  and  do  not  enter  the  general  circulation 
unless  fat  is  present  in  the  intestine.  The  blood  of  Sudan-stained 
animals,  under  normal  conditions  of  feeding,  is  never  free  from  the 
fat  stain.  The  dye  put  out  in  the  biliary  secretion  is  reabsorbed 
in  the  digesting  fat,  and  a  continuous  circulation  from  gut  to  blood, 
and  return  is  established.  The  elimination  of  the  stain  from  the 
circulation,  when  all  possibility  of  reabsorption  is  removed,  takes 
place  slowly.  The  stained  fat  was  found  in  the  blood  of  a  cat 
nine  and  one-half  hours  after  it  had  been  injected  into  the  femoral 
vein. 

FAT  TRANSPORT  IN  STARVATION  AND  PATHOLOGICAL  CONDITIONS: 
PHOSPHORUS  AND  PHLORHIZIN  POISONING. 

We  have  attempted  to  follow  the  migrations  of  Sudan-stained 
fats  under  conditions  in  which  a  transport  of  fat  is  well-known  to 
occur,  namely,  in  starvation  and  after  poisoning  with  phosphorus 
or  phlorhizin.  The  experimental  animals  were  fed  in  advance 
for  a  period  of  three  to  five  weeks  on  Sudan-stained  food.  Phos- 
phorus was  administered  subcutaneously,  dissolved  in  oil;  phlor- 
hizin similarly  in  solution  in  sodium  carbonate.  Other  details 
of  selected  protocols  are  summarized  in  tabular  form: 


Sudan  III  in  pathological  fat  transport  and  starvation. 


ANIMAL 

DURATION 
OF 
EXPERI- 
MENT 

FAT 
CONTENT  OF 
LIVER 

STAIN  IN 

Bile 

Blood 

days 

per  cent 

Cat  I,  13 

5 

56.0 

Present 

Present 

Starvation  j 

Cat  I,  18 

5 

33.5 

Present 

Present 

Guinea  pig  

1 

7.9 

Present 

Phosphorus  J 

Cat  XI,  23...  . 

9 

64.3 

Present 

Present 

Hen  X,  20 

4 

59.0 

Present 

poisoning  1 

Hen  XI,  29 .  .  . 

19 

40.5 

Present 

Phlorhizin  j 

Cat  XII,  6. . .  . 

12 

15.8 

Present 

Present 

poisoning  \ 

Cat  XI,  19. . .  . 

7  . 

11.1 

Present 

Present 

Lafayette  B.  Mendel  and  Amy  L.  Daniels  91 


Neither  in  the  foregoing  nor  in  numerous  other  comparable 
experiments  in  which  a  transport  of  fat  (fatty  infiltration)  was 
induced,  was  any  evidence  obtainable  of  dye  in  the  extracts  of  the 
liver  tissue  or  in  frozen  sections  thereof.  The  constant  finding  of 
the  Sudan  III  in  both  the  blood  and  bile  makes  it  evident  that 
the  dye  migrates  from  the  stained  adipose  tissue  and  is  brought 
to  the  liver  where  it  is  eliminated  in  the  bile.  The  observations 
give  an  additional  indication  that  the  fatty  livers  in  these  patho- 
logical conditions  are  produced  by  infiltration  of  fat;  for  it  is  diffi- 
cult to  believe  that,  if  the  high  content  of  liver  fat  had  been  ob- 
tained by  a  degeneration  process  in  the  hepatic  tissue,  such  an 
accumulation  of  dye  in  the  bile  would  have  taken  place. 

FAT  TRANSPORT  TO  THE  EMBRYO. 

The  question  of  the  origin  of  foetal  fat  has  been  much  debated;22 
It  involves  the  broader  problem  of  the  passage  of  substances 
through  the  placental  barrier.  S.  H.  and  S.  P.  Gage  ('08)  failed 
to  find  the  adipose  tissues  of  the  young  stained,  when  stained  fats 
were  fed  to  pregnant  mothers.  Hofbauer  ('05)  believed  that  he 
found  particles  of  dye  in  the  foetal  blood  and  assumed  that  they 
had  become  separated  from  fat  metabolized  by  the  embryo.  His 
method — microscopic  examination — is  scarcely  adapted  to  deter- 
mine this  point,  however. 

Numerous  experiments  in  which  we  have  fed  rats  and  cats  with 
Sudan-stained  food  or  Biebrich  Scarlet  throughout  the  period 
of  gestation  have  uniformly  shown  an  absence  of  the  dye  in  the 
foetus  or  the  newly-born  young.  Two  illustrative  protocols,  selected 
from  many  similar  ones,  will  suffice  to  show  our  method  of  inves- 
tigation. 

Rat  C.  Sudan-feeding  was  begun  sixteen  days  before  the  young  were 
born.  The  alimentary  tract  was  removed  from  one  of  them  soon  after 
birth.  Its  contents  were  distinctly  pink  (from  mothers'  milk).  The  ether 
extract  of  the  entire  residual  body  was  uncolored.  Subsequent  examina- 
tion of  the  mother  showed  deeply  stained  adipose  tissue. 


22  Cf.  Ahlfeld:  Centralbl.  f.  Gynaekol,  i,  p.  265,  1877;  Thiemich:  Cen- 
tralbl.  f.  Physiol,  xii,  p.  850,  1898;  Jahrb.  f.  Kinderheilk.,  lxi,  p.  174,  1905; 
Hofbauer,  J.:  Arch.  f.  Gynaekol.,  lxxvii,  p.  139,  1906;  Oshima:  Zentralbl. 
f.  Physiol.,  xxi,  p.  297,  1907;  Bondi:  Arch.  f.  Gynaekol.,' xciii,  p.  189,  1911. 


92  Behavior  of  Fat-Soluble  Dyes 


Cat  B.  Was  fed  80  ragms.  of  Sudan  III  every  second  day  for  eighteen 
days  prior  to  birth  of  kittens.  Aside  from  the  stomach  contents  there  was 
no  pink  in  the  ether  extract  of  tissues  of  the  young  examined  soon  after 
birth.    The  adipose  tissue  of  the  mother  was  deeply  stained. 

Although  it  is  unlikely  from  such  findings  that  stained  fat  can 
pass  through  the  placenta,  this  is  not  necessarily  conclusive  evidence 
that  the  foetal  fat  has  its  origin  in  substances  other  than  fat.  The 
findings  in  the  case  of  the  alimentary  epithelial  tissues  and  glandu- 
lar structures  however  add  little  likelihood  to  the  transport  or 
deposition  of  the  fat  in  a  non-stainable  combination. 

FAT  TRANSPORT  INTO  MILK. 

The  precise  relation  of  milk  fat  to  food  fat  and  the  extent  to 
which  the  latter  can  pass  directly  into  the  mammary  secretion 
without  first  becoming  a  part  of  the  body  stores  is  not  easily  deter- 
mined. S.  H.  and  S.  P.  Gage  ('09)  found  Sudan  III  in  the  milk 
of  rats  after  prolonged  feeding  with  the  dye;  this,  however,  is  no 
proof  of  the  immediate  origin  of  the  milk  fat  from  the  food,  since 
the  fat  depots  of  the  rats  were  also  stained.  In  explanation  of  the 
observations  that  foreign  food  fats  have  more  frequently  been 
found  secreted  in  the  milk  of  smaller  animals  (goats,  sheep,  dogs, 
rats)  than  of  cows,  it  has  been  suggested  that  the  milk  secretion  is 
more  directly  dependent  upon  the  food  supply  in  the  smaller 
species.2"  However,  the  marked  differences  in  the  time  required 
to  stain  the  adipose  tissue  of  guinea  pigs  and  rabbits  with  Sudan 
III  in  comparison  with  cats  and  rats,  suggests  that  the  discrep- 
ancies noted  above  may  bear  some  relation  to  the  readiness  with 
which  the  different  animals  absorb  and  store  fat. 

Experimental.  We  have  investigated  the  appearance  of 
Sudan  III  in  the  milk  after  feeding  the  dye  both  before  and  during 
the  period  of  lactation.  When  animals,  notably  cats,  have  refused 
to  eat  stained  fat,  the  dye  has  been  administered  in  capsules  either 
directly  before  or  after  a  meal  rich  in  fat.  This  fact  is  important 
for  successful  results.  In  the  case  of  cats  and  rats  the  character 
of  the  milk  was  determined  by  examining  the  stomach  contents 
of  suckling  young.    Needless  to  say  great  care  must  be  taken  to 

23  Cf.  Lusk:  Science  of  Nutrition,  1909,  p.  237. 


Lafayette  B.  Mendel  and  Amy  L.  Daniels  93 


have  the  cages  scrupulously  free  from  stained  food  which  might 
lead  to  erroneous  conclusions. 

It  is  scarcely  necessary  to  repeat  here  the  details  of  the  many 
trials,  since  the  methods  are  fairly  obvious.  Both  Sudan  III  and 
Biebrich  Scarlet  were  found  to  be  secreted  into  the  milk  by  rats; 
Sudan  excretion  was  likewise  observed  in  cats,  guinea  pigs  and 
a  goat.  In  the  case  of  the  goat,  one  gram  of  Sudan  III  dissolved 
in  oil  was  added  to  the  feed  twice  daily  during  six  successive  days.24 
The  milk  drawn  nine  hours  after  the  first  dose  showed  the  presence 
of  the  dye,  the  tint  increasing  with  the  subsequent  milkings.'  The 
guinea  pigs  received  2  cc.  of  stained  olive  oil  every  other  day. 

An  important  fact  in  this  connection  is  the  observation  that 
the  color  disappears  from  the  milk  when  the  Sudan-feeding  is 
discontinued,  despite  the  persistence  of  the  stain  in  the  adipose 
tissues  of  the  secreting  animals.  This  was  likewise  true  in  exper- 
iments with  Biebrich  Scarlet. 

The  following  protocol  illustrates  the  transport  of  storage  stained 
fat  during  starvation  and  the  passage  of  the  dye  into  the  milk: 

Rat  11.  Was  fed  Sudan-stained  food  during  the  period  of  gestation. 
Soon  after  the  birth  of  the  young,  April  30,  the  cage  was  cleaned  and  un- 
stained food  thenceforth  employed.  On  May  14  the  ether  extract  of  milk 
found  in  the  stomach  of  a  suckling  rat  was  uncolored.  The  mother  was 
now  starved  two  days.  At  the  end  of  this  time  the  milk  in  the  stomach  of 
another  one  of  the  young  gave  a  faintly  pink  ether  extract.  The  adipose 
tissue  of  the  adult  was  found  to  be  stained  still.  This  experiment  was 
duplicated  with  another  mother. 

Like  S.  H.  and  S.  P.  Gage,  we  have  failed  to  induce  the  secretion 
of  Sudan  III  in  the  milk  of  cows.  A  Holstein  cow  was  given  7.5 
grams,  twice  daily,  dissolved  in  olive  oil  and  added  to  the  mash 
feed  on  three  successive  days,  without  positive  results.  In  con- 
sidering this  we  recall  that  the  milk  of  the  goat  and  guinea  pig — 
animals  in  whose  diet  fat  likewise  plays  a  comparatively  small  role 
— was  decidedly  faint  in  color  in  comparison  with  the  milk  of  cats 
and  rats.  Bearing  in  mind  the  necessity  of  fat  for  the  transport 
of  the  dye  an  explanation  at  once  suggests  itself  for  the  inequalities 
here  observed. 

24  This  experiment  was  conducted  at  the  New  York  Agricultural  Ex- 
periment Station  in  Geneva,  through  the  kindness  of  Director  W.  H.  Jor- 
dan. 


94  Behavior  of  Fat-Soluble  Dyes 


SUMMARY. 

Some  of  the  fat-soluble  dyes,  introduced  into  the  organism  by 
various  paths,  are  deposited  in  the  adipose  tissues  and  bone  marrow. 
The  renal  and  nervous  tissues  are  free  from  the  stain,  even  when 
the  fatty  tissues  are  deeply  colored.  Muscle  probably  does  not 
take  up  the  dye.  It  is  seldom  found  in  the  liver,  because  the  fat- 
soluble  dyes,  which  are  insoluble  in  water,  dissolve  readily  in  the 
bile  and  are  excreted  thereby  into  the  intestine  from  which  they 
can  be  reabsorbed. 

The  fat-soluble  dyes  may  enter  the  organism  from  the  alimentary 
tract  through  the  lymphatics,  in  solution  in  fat;  or  by  the  portal 
circulation,  dissolved  in  reabsorbed  bile.  They  do  not  pass  beyond 
the  liver  unless  fat  is  present  to  transport  them.  Then  they  may 
be  found  in  the  blood,  which  is  rarely  free  from  the  dye  in  a  nor- 
mally fed  animal  that  has  once  been  stained.  A  cycle  between 
intestine,  bile  and  blood  becomes  established.  No  elimination  of 
the  dyes  occurs  through  the  kidneys,  except  when  an  alimentary 
lipuria  arises  (in  rabbits  and  rats). 

Contrary  to  the  assertion  of  others,  the  stained  fat  is  no  less 
available  to  the  organism  than  the  unstained. 

In  cases  conducive  to  fat  transport — in  starvation,  phosphorus  - 
and  phlorhizin-poisoning — stained  fat  migrates  from  the  stained 
depots  to  the  blood  and  the  liver  cells.  Here  the  dye  is  separated 
and  secreted  into  the  bile;  so  that  the  liver,  though  having  a  high 
content  of  fat,  may  be  free  from  the  dye. 

Stained  fat  does  not  traverse  the  placenta.  The  blood  of  the 
foetus  and  the  fat  of  young  born  of  Sudan-stained  mothers  is  free 
from  dye. 

The  excretion  of  Sudan  III  and  Biebrich  Scarlet  in  milk,  when 
they  are  given  with  food  fat,  suggests  that  the  latter  may  pass 
directly  into  the  mammary  secretion.  With  cats  and  rats  the 
results  are  striking,  but  the  dye  excretion  in  milk  ceases  when  the 
stained  food  is  no  longer  fed.  In  guinea  pigs  and  goats  the  secre- 
tion of  dye  in  the  milk  is  positive;  in  the  cow  it  has  not  yet  been 
demonstrated.  The  variation  in  the  outcome  in  the  different 
species  may  be  due  to  variations  in  the  relative  abundance  In  the 
dietaries  of  fat  necessary  for  the  absorption  and  transport  of  the 
dye.    This  explanation  is  emphasized  by  the  observation  that  those 


Lafayette  B.  Mendel  and  Amy  L.  Daniels  95 


animals  (cats,  rats,  hens,  pigeons)  for  which  fat  enters  more  largely 
into  the  diet,  become  stained  more  easily  or  speedily  than  animals 
which  are  accustomed  to  ingest  relatively  smaller  amounts  of  fat. 

BIBLIOGRAPHY  OF  EXPERIMENTS  WITH  SUDAN  III  AND  OTHER 
FAT-SOLUBLE  DYES. 

Biedermann:  Arch.  f.  d.  ges.  Physiol.,  lxxii,  p.  105,  1898. 

Bondi  and  Neumann:  Zentralbl.  f.  Biochem.  u.  Biophysik,  x,  1453,  1910. 

Daddi:  Arch.  ital.  de  biol.,  xxvi,  p.  142,  1896. 

Fischer:  Centralbl.  f.  allgemeine  Pathol,  u.  pathol.  Anat.,  xiii,  p.  943, 1902. 

Franz  and  Von  Stejskal:  Zeitschr.  f.  Heilkunde,  xxiii,  p.  441,  1902. 

Gage,  S.  H.  and  S.  P.:  Science,  xxviii,  p.  494,  1908. 

Gage,  S.  H.  and  S.  P.:  Anat.  Rec,  iii,  1909. 

Hofbauer  L. :  Arch.  f.  d.  ges.  Physiol.,  lxxxi,  p.  263,  1900. 

Hofbauer  I.:  Grundziige  einer  Biologic  der  menschlichen  Placenta  mitbe- 
sonderer  Beriiksichtigung  der  Fragen  der  fdtalen  Erndhrung,  Wien  und  Leip- 
zig, 1905. 

Jacobsthal:  Verhandl.  d.  deutsch.  pathol.  Gesellsch,  xiii,  p.  380,  1909. 
Mann:  Physiological  Histology,  p.  306-07,  1902. 
Mendel:  Amer.  Journ.  of  Physiol.,  xxiv,  p.  493,  1909. 
Michaelis:  Deutsch.  med.,  Wochenschr.,  xxvii,  p.  183,  1901. 
Neisser  and  Braeuning:  Zeitschr.  f.  exper.  Pathol,  u.  Therap.,  iv,  p. 
747,  1907. 

Pfluger:  Arch.  f.  d.  ges.  Physiol.,  lxxxi,  p.  375,  1900. 
Riddle:  Science,  xxvii,  p.  945,  1908. 
Riddle:  Journ.  of  Exper.  Zobl.,  viii,  p.  163,  1910. 
Sitowski:  Anz.  d.  Akad.  d.  Wissensch.  in  Krakau,  p.  542,  1905. 
Staniewicz:  Zentralbl.  f.  Biochem.  u.  Biophysik.,  x,  1435,  1910. 
Whitehead:  Arrier.  Journ.  of  Physiol.,  xxiv,  p.  294,  1909;  xxv,  p.  xxviii, 
1909-10. 


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PHYSIOLOGISCHE  CHEMIE 

unter  Mitwirkung  von 

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furt  a.  M.,  A.  ELLINGER-Konigsberg,  H.  EULER-Stockholm,  EMIL 
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R.  v.  ZEYNEK-Prag 

herausgegeben  von 

A.  KOSSEL, 

Professor  der  Physiologie  in  Heidelberg. 


Beobachtungen  iiber  Wachstum  bei  Futterungsversuchen 
mit  isolierten  Nahrungssubstanzen. 

Von 

Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 
unter  Mitwirkung  von  Edna  L.  Ferry. 

Mit  65  Kurvenzeichnungen  im  Text. 


Separat-Abdruck  aus  Band  80,  Heft  5, 

STRASSBURG 
VERLAG  von  KARL  J.  TRUBNER. 
1912. 


ACHTZIGSTER  BAND,  FUNFTES  HEFT. 


In  halt.  Seite 

Osborne,  Thomas  B.,  Lafayette  B.  Mendel  und  Edna  L.  Ferry. 

Beobachtungen  uber  Wachstum  bei  Futterungsversuchen 
mit  isolierten  Nahrungssubstanzen.    Mit  65  Kurvenzeich- 

nungen  im  Text    .  .     307 

Rogozinski,  F.    Zur  Methylierung  des  Glupeins   371 

Pringsheim,  Hans.  Uber  den  fermentativen  Abbau  der  Hemi- 
cellulosen.  I.  Mitteilung.  Ein  Trisaccharid  als  Zwischen- 
produkt  der  Hydrolyse  eines  Mannans   376 


Fiir  die  nachsten  Hefte  sind  Arbeiten  eingegangen  von: 

L.  Wacker,  G.  Trier,  E.  Abderhalden  und  A.  Eodor,  e.  Letsche, 
G.  Th.  Morner,  A.  DorneiyP.  Rohland. 


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Beobachtungen  uber  Wachstum  bei  Futterungsversuchen 
mit  isolierten  Nahrungssubstanzen. 1 ) 

Von 

Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 
unter  Mitwirkung  von  Edna  L.  Ferry. 

Mit  65  Kurvenzeichnungen  im  Text. 


(Aus  dem  Laboratorium  der  Connecticut  Agricultural  Experiment  Station  und  dem 
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Inhalt. 

Einleitung.  Veranderungen  des  Wachstums,  die  durch  aufterhalb  der 
Ernahrung  liegende  Faktoren  oder  durch  ungeniigende  Nahrungszufuhr 
verursacht  werden.  Anderungen  des  Wachstums  durch  qualitativ  (che- 
misch)  ungeeignete  Nahrungszufuhr.  Eiweifi  und  Wachstum.  Warum 
wachsen  die  Tiere  bei  gewissen  Ernahrungsformen  nicht?  Eiweifrkorper  der 
Leguminosen  und  Wachstum.  Quantitative  Gesichtspunkte  uber  Wachstums- 
hemmung.  Kunstliche  Salzmischungen  und  Wachstum.  Wachstum  bei 
einer  von  atherloslichen  Substanzen  freien  Diat.  Die  Unterdriickung  des 
Wachstums  und  die  Fahigkeit,  das  Wachstum  wieder  aufzunehmen.  Einige 
Bemerkungen  und  SchluMolgerungen. 

Einleitung. 

Die  vorliegenden  Beobachtungen  setzen  voraus,  daB 
die  Wachstumszunahme  der  jungen  weiBen  Ratte  durch  eine 
charakteristische  Kurve  ausgedriickt  werden  kann.  Die  Auf- 
merksamkeit  war  deshalb  besonders  darauf  gerichtet,  die  Fak- 
toren, welche  das  normale  Wachstum  hemmen  oder  vollstandig 
behindern,  zu  bestimmen. 

Welche  Ernahrungskomponenten  sind  fur  eine  ange- 
messene  Entwicklung  unentbehrlich  ?  In  welchen  Quantitaten 
mussen  sie  an  die  Tiere  verfuttert  werden?  Kann  nach  ge- 
hemmtem  oder  unterdriicktem  Wachstum  eine  Wiederherstel- 

l)  Die  Ausgaben  fur  diese  Untersuchung  trug  die  Connecticut 
Agricultural  Experiment  Station  und  die  Carnegie  Institution  of  Wash- 
ington, D.  C. 

Hoppe-Seyler's  Zeitschrift  f.  physiol.  Chemie.  LXXX.  21 


308        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 

lung  erwartet  werden  und  wenn  so,  wie?  Gibt  es  feststehende 
qualitative  Unterschiede  zwischen  der  zur  Erhaltung  und  der 
zum  Wachstum  notigen  Ernahrung?  Dies  sind  einige  Probleme, 
die  der  Losung  harren.  Und  es  wird  sich  zeigen,  daB  das 
Studium  der  feststellbaren  Wachstumshemmung,  einige  wenige 
Falle  ausgenommen,  die  Formulierung  positiver  Ergebnisse 
moglich  gemacht  hat. 

Die  vorliegende  Arbeit  beriehtet  in  ausgedehnter  Weise 
iiber  schon  friiher  an  weiBen  Ratten  vorgenommene  Fiitterungs- 
versuche,  in  welchen  die  Feststellung  einer  geeigneten  Er- 
nahrung mit  Mischungen  einzelner  Nahrungsstoffe  angestrebt 
wurde.  Unsere  friiheren  Experimente  haben  wir  gleichzeitig 
mit  der  angegebenen  Literatur  sowie  den  Methoden  der  Ge- 
fangenhaltung,  Futterung  und  Pflege  der  Tiere  anderweitig 
bereits  veroffentlicht.1)  Aus  den  bereits  veroffentlichten  Fest- 
stellungen  wollen  wir  nur  wiederholen,  daB,  verschiedentlich 
debattierte  Schwierigkeiten  des  Versuchsplans  anlangend,  die 
Einformigkeit  der  Ernahrung  als  kein  ernstliches  Hindernis  sich 
erwiesen  hat.  Aufzeichnungen  iiber  in  Perioden  vonl— 2Jahren 
mit  unveranderter  Kost  ernahrten  Ratten  lassen  iiber  diese 
Tatsache  keinen  Zweifel  aufkommen.  Als  Bestatigung  dienen 
die  Ver such e  von  Hart,  Mc  Gollum,  Steenbock  und  Hum- 
phrey, welche  durch  3  Jahre  eine  im  wesentlichen  unveran- 
derte  Kost  an  ihre  Tiere  verfiittert  haben  und  zu  dem  Schlusse 
kamen,  daB  die  Einformigkeit  der  Ernahrung  kein  storender 
Faktor  und  bei  den  Futterungsversuchen  keineswegs  von  solcher 
Tragweite  ist,  wie  gewohnlich  behauptet  wird.2)  Ferner  er- 
reichen  weiBe  Ratten  trotz  unserer  Gefangenhaltung  und  der 
begrenzten  Freiheit  in  Bewegung  und  Leben  ein  verhaltnis- 

*)  T.  B.  Osborne  und  L.  B.  Mendel,  Futterungsexperimente  mit 
isolierten  Nahrungssubstanzen,  Carnegie  Institution  of  Washington,  Publi- 
cation 156,  Parts  I  and  II,  1911;  Science  N.  S.,  1911,  Bd.  34,  S.  722—732, 
und  Zeitschrift  fur  biologische  Technik  und  Methodik,  1912,  Bd.  2,  S.  313—318. 

8)  E.B.Hart,  E.  V. Mc.  Collum,  H. Steenbock  u.  G. C.Humphrey, 
Physiological  Effect  on  Growth  and  Reproduction  of  Rations  Balanced 
from  Restricted  Sources.  Univ.  of  Wisconsin  Agri.  Expt.  Station,  Research 
Bulletin  Nr.  17,  June  1911,  p.  131—205;  cf.  also  Journal  of  Biological 
Chemistry,  1912,  Bd.  11,  S.  XII. 


Uber  FiUterungsversuchen  mit  isolicrten  Nahrungssubstanzen.  309 

maBig  hohes  Alter  im  Vergleich  zu  Tieren,  die  in  dieser  Be- 
ziehung  weniger  Einschriinkung  erleiden  mussen,  eine  Beobach- 
tung,  in  der  wir  uns  mit  der  jiingst  veroffentlichten  Ansicht 
von  Slonaker1)  in  Einklang  befinden.  Die  GroBe  des  Wachs- 
tums,  gemessen  durch  den  Wechsel  des  Korpergewichts  und 
graphisch  aufgezeichnet,  ist  noch  immer  ein  durchaus  ge- 
niigender  Index  fur  die  Schwankungen,  mit  welchen  wir  uns 
beschaftigen.  Die  gut  bekannten  Kurven  von  Donaldson2) 
uber  das  wechselnde  Korpergewicht  der  Albinoratte  sind  durch 
Slonakers  Messungen  bestatigt  worden;3)  diesen  konnen  wir 
nunmehr  eine  betrachtlich  groBere  Anzahl  von  eigenen  hinzu- 
fugen.   Die  Resultate  werden  in  Kurve  1  gezeigt. 

Kurve  1  zeigt  die  durchschnittlichen  Schwankungen  im 
Korpergewicht  der  mannlichen  und  weiblichen  Albinoratte,  wie 
sie  durch  Bestimmungen  von  Donaldson,  Slonaker  und 
Osborne  und  Mendel  festgestellt  worden  sind. 

Es  wird  sich  zeigen,  daB  unsere  Ratten  aus  Griinden,  die 
wir  noch  nicht  genugend  haben  bestimmen  konnen,  die  Neigung 
zeigen,  etwas  kleiner  als  die  von  Donaldson  zu  bleiben.  Dies 
muB  beim  Studium  unserer  Wachstumsdaten  im  Auge  be- 
halten  werden. 

Veranderungen  des  Wachstums,  die  durch  aufierhalb  der  Ernahrung 
liegende  Faktoren  oder  durch  ungemigende  Nahrungszufuhr 
verursacht  werden. 

Gewisse  Faktoren  bedingen  so  ersichtlich  eine  Hemmung 
des  Wachstums,  daB  sie  keiner  ausfuhrlichen  Besprechung  be- 
diirfen.  Bekannt  ist  dies  von  MiBhandlungen,  angeborenen 
Defekten  und  Krankheiten,  mogen  sie  nun  familienweise  z.  B. 

*)  J.  R.  Slonaker,  Die  normale  Lebensfahigkeit  der  Albinoratte 
von  ihrer  Geburt  bis  zu  ihrem  normalen  Tod,  ihre  Wachstumszunahme 
und  Lebensdauer.    Journal  of  Animal  Behavior,  1912,  Bd.  2,  S.  20—42. 

2)  H.  H.  Donaldson,  Ein  Vergleich  der  weifien  Ratte  mit  dem 
Menschen  in  bezug  auf  Wachstum  des  ganzen  Korpers.    Boas  Memorial 
Volume,  New  York,  1906.    Cf.  also  Osborne  and  Mendel,  Carnegie 
Institution  of  Washington,  Publication  156,  Part  I,  S.  14,  Part  II,  S.  87. 
s)  J.  R.  Slonaker,  Journal  of  Animal  Behavior,  1912,  Bd.2,S.20-42. 

21* 


310        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Kurve  1. 


Uber  Fiitterungsversuche  mit  isolierten  Nahrungssubstanzcn.  311 

als  Infektion  usw.  oder  auch  in  mehr  kryptogener  Form  auftreten. *) 
Eine  andere  wichtige  Hemmung  des  normalen  Wachstums 
bildet  die  Unzulanglichkeit  im  Energieersatz,2)  obwohl  die 
Wachstumshemmung  keineswegs  immer  mit  Gewichtsstillstand 
zusammenfallt. 3)  Dies  sind  Erscheinungen,  welche  in  den  vor- 
liegenden  Untersuchungen  nicht  Gegenstand  spezieller  Studien 
sein  konnen.  Die  Moglichkeiten,  welche  sie  darstellen,  miissen, 
so  besonders  der  Eintritt  von  Krankheiten,  als  Gelegenheiten 
zur  Gewichtsabnahme  betrachtet  werden,  die  keineswegs  der 
Diat  allein  zuzuschreiben  sind.  So  muB  also  ein  Unterschied 
gemacht  werden  zwischen  aktueller  Herabsetzung  des  Er- 
nahrungszustandes  (Sinken  des  Korpergewiehts)  und  Unter- 
druckung  des  Wachstums  bei  gleichbleibendem  Korpergewicht 
in  Zeiten,  in  welchen  ein  normales  Wachstum  erwartet  werden 
konnte. 

Anderungen  des  Wachstums  durch  qualitativ  (chemisch)  ungeeignete 

Nahrungszufuhr. 

In  unseren  friiheren  Studien  haben  wir  bereits  beobachtet, 
daB  Tiere  in  ersichtlich  geniigendem  Ernahrungszustande  und 
gleichem  Korpergewicht  und  -maB  verbleiben  konnen,  wenn  sie 
sich  in  einem  Alter  befinden,  in  dem  ein  kraftiges  Wachstum 
ohnehin  zu  erwarten  ist.  Obgleich  bei  diesen  Tieren  wahrend 
langer  Perioden  keine  sichtbare  Abnahme  oder  sonstige  Anzeichen 
von  Gesundheitsstorungen  bestanden,  wuchsen  sie  aber  nicht 
in  einem  MaBe,  das  dem  normalen  entsprochen  hatte.  Solche 
Falle  zeigen,  was  wir  Gleichgewicht  ohne  Wachstum  nennen. 

*)  Manche  Debatten  uber  diese  Fragen  finden  sich  in  der  Literatur 
uber  Kinderheilkunde.  Gf.  E.  Schloss,  Die  Pathologie  des  Wachstums 
im  Sauglingsalter,  Berlin  1911. 

8)  J.  Rosens  tern,  Uber  Inanition  im  Sauglingsalter.  Ergebnisse 
der  inneren  Medizin  und  Kinderheilkunde,  1911,  Bd.  7,  S.  332. 

3)  H.  Aron,  Biochemische  Zeitschrift,  1910,  Bd.  30,  S.  207;  Phi- 
lippine Journal  of  Science  (B),  1911,  Bd.  6,  Nr.  1,  S.  1—50;  H.  J.  Waters, 
Das  Bestreben  der  Tiere,  unter  verschiedenen  Bedingungen  zu  wachsen. 
Proceedings  Society  for  the  Promotion  of  Agricultural  Science,  1908, 
Bd.  29,  S.  3. 


312        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 

Entweder  die  Nahrung  ermangelte  einiger  spezifischer  che- 
mischer  Substanzen,  die  zur  Bildung  neuer  Gewebe  oder  zum 
Wachstum  notig  waren,  oder  das  beziigliche  Verhaltnis  der 
einzelnen  Bestandteile  der  Nahrung  zueinander  war  nicht  das 
richtige.  Unsere  Nahrungsgemische  bestanden  zu  dieser  Zeit 
aus  einzelnen  EiweiBkorpern, x)  Fett,  Starke  und  Zucker,  und 
einer  Salzmischung  nach  Angaben  von  Rohmann2)  welch 
letztere  folgende  Zusammensetzung  hatte: 


Salzmischung  I. 


Ca3(P04)2 

10,0  g 

K2HP04 

37,0  » 

NaCl 

20,0  » 

Na-Gitrat 

15,0  » 

Mg-Citrat 

8,0  » 

Ca-Lactat 

8,0  » 

Fe-Citrat 

2,0  » 

100,0  g. 

Seit  der  spater  folgende  Versuch  gezeigt  hat,  daB  ein 
normales  Wachstum  erfolgt,  wenn  der  Zucker  durch  Lactose, 
die  hier  angewandten  Salze  durch  eine  veranderte  Mischung 
ersetzt  werden,  sofern  die  iibrigen  Nahrungsbestandteile  in  bezug 
auf  Quantitat  und  Qualitat  unverandert  bleiben  (S.  49),  miissen 
wir  die  folgende  erste  Versuchsreihe  als  ein  Beispiel  von 
Wachstumsunterdruckung  ohne  merklichen  Verfall  betrachten 

*)  Es  mufi  bemerkt  werden,  daft  auf  die  Darstellung  der  bei  diesen 
Untersuchungen  angewandten  Eiweiftkorper  die  groftte  Sorgfalt  angewandt 
wurde.  Die  Produkte  waren  so  rein,  als  man  sie  fur  den  Zweck  einer 
genauen  Eiweifianalyse  nur  erwarten  konnte.  Die  Notwendigkeit  einer 
Genauigkeit  in  dieser  Richtung  kann  nicht  streng  genug  betont  werden, 
seit,  wie  wir  gezeigt  haben,  kleine  Beimischungen  entschieden  die  Re- 
sultate  eines  Fiitterungsversuchs  andern.  Cf.  Carnegie  Institution  of  Wash- 
ington, Publication  156,  Part  II,  1911,  S.  84. 

2)  Rohmann,  Allgemeine  med.  Zentralzeitung,  1903,  Nr.  1;  1908, 
Nr.  9.  Cf.  Malys  Jahresbericht,  1903,  Bd.  33,  S.  823;  1908,  Bd.  38,  S.  659. 
Die  Griinde  fur  den  Gebrauch  dieser  Mischung  sind  in  unseren  ersten 
Berichten  angegeben,  Publication  156,  Carnegie  Institution  of  Washington, 
Part  I,  1911,  S.  32. 


Uber  Ftttterungsversuche  mil  isolierten  Nahrungssubstanzen.  313 

und  sie  den  Nicht-EiweiB-  und  Nicht-Fettbestandteilen  der 
Nahrung  zuschreiben.  Die  folgenden  Kurven  2  und  3  iilu- 
strieren  dies.1) 


7   

1 

/ 

/ 

s 

1  

|   

i  

f 



/ 

/ 

/ 

/ 

/ 

-> 

4^  ^  /<5tf 


s 

/ 

s 

/ 

/ 

/ 

/ 

— /« — 
/ 

/ 

/ 

/ 

/ 

— ^  

?.5<?//7__j 

0  20  <5^?  <^£7  /^£7 

Tage 


Kurve  2. 


Kurve  3. 


Kurve  2  (Ratte  100  d")  zeigt  eine  Unterdriickung  des 
Wachstums,  die  den  Nicht-Eiweifl-Faktoren  der  Diat  zuzu- 
schreiben  ist. 


*)  In  alien  unseren  Kurven  bezeichnen  die  Abszissen  die  Tage  und 
die  Ordinaten  das  gegenwartige  Korpergewicht  (ununterbrochene  Linie) 
Oder  die  Nahrungszufuhr  (punktierte  Linie)  in  Gramm.  In  einigen  Ta- 
bellen  ist  die  durchschnittliche  (normale)  Wachstumsgrofte  der  Tiere  des- 
selben  Geschlechts  (siehe  Kurve  I)  zum  Vergleich  durch  eine  gebrochene 
Linie  gekennzeichnet.  Die  Kurve  fur  Nahrungszufuhr  zeigt  die  Quantitat 
der  in  einer  Woche  verzehrten  Nahrung.  Die  Zahlen  auf  den  Kurven 
kennzeichnen  den  Wechsel  der  Ernahrungsweise  und  korrespondieren  mit 
den  Angaben  in  den  Tabellen  uber  die  Zusammensetzung  der  Nahrung. 


Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Nahrung: 

0/0 

Glutenin  (Weizen)  18,0 
Starke  14,5-34,5 
Zucker  15,0-20,0 
Agar  5,0 
Salzmischung  I  2,5 
Fett  45,0-20,0 

Kurve3  (Ratte  191  </)  zeigt  eine  Unterdriickung  des 
Wachstums,  welche  den  Nicht-EiweiB-Faktoren  der  Diat  zu- 
geschrieben  werden  muB. 


Nahrung: 


°/o 

Casein  (Kuh) 

18,0 

Starke 

32,5 

Zucker 

20,0—17,0 

Agar 

5,0 

Salzmischung  I 

2,5 

Fett 

22,0—25,0 

Unsere  Versuche  haben  gezeigt,  daB  bei  jungen  Ratten 
durch  eine  Nahrungszufuhr  von  Milchpulver  und  Fett  eine  Ge- 
wichtszunahme  erzeugt  wird,  wie  die  folgenden  Kurven  sie 
veranschaulichen. 

Kurve  4  (Ratte 
96  ?)  und  Kurve  5 
(Ratte  97  ?)  zeigen 
eine  entsprechende 
Wachstumszunahme 
bei  einer  Diat,  die 
aus  einem,  alle  festen 

Restandteile  der 
Milch  enthaltenden 
Pulver  besteht,  und 
zwar  in  der  Form 
eines  Handelsprapa- 
rates,  das  als  «Tru- 
milk»  bekannt  ist. 
Weitere  Reispiele 


0  20         W         60  SO  100 


Kurve  4. 


Uber  FiUterungsversuchc  mit  isolicrten  Nahrungssubstanzen.  315 


160 


siehe:  «Fiitterungs- 
experimente  mit  iso- 
lierten  Futtersub- 
stanzen».  Carnegie 
Institution  of  Wash-  fto 
ington,  Publication 
156,  Part  II,  Charts 
XXVIII,  XXIX,  XXXI, 
XXXII. 


N ah rung 

Milchpulver 
Starke 

Salzmischung  I 
Fett 


60,0 
15,7 
1,0 
23,3 


Kurve  5. 

Die  Forschung  iiber  die  Rolle  der  anorganischen  Elemente 
im  Hinblick  auf  diesen  Wachstumsstillstand  schien  zunachst 
mit  uniiberwindlichen  Schwierigkeiten  verbunden  zu  sein.  Die 
theoretischen  Moglichkeiten  sind  mannigfach  und  kompliziert. 
Ein  neuerer  Forscher  meint:  «Damit  stehen  wir  vor  einem 
unerschopflichen  Gebiete  der  Experimentalforschung,  in  welchem 
zurzeit  nur  die  ersten  Ansatze  zur  Gewinnung  allgemeiner  Ge- 
sichtspunkte  gemacht  sind.*1)  Nach  zahlreichen  Fehlschlagen 
mit  der  kiinstlichen  Diat  und  Anderung  sowohl  der  relativen 
Verhaltnisse  der  verschiedenen  Ionen  als  auch  des  Charakters  der 
Kohlenhydrate  und  Fette  trat  der  Fall  ein,  daB  der  eiweiB-  und 
fettfreie  Bestandteil  der  Milch  den  Fingerzeig  zu  einer  erfolg- 
reichen  Fiitterung  mit  solchen  EiweiBkorpern  gab,  welche  bei 
Unterdriickung  des  Wachstums  nicht  als  unwirksamer  bezw.  hem- 
mender  Faktor  in  Betracht  zu  kommen  scheinen.  Die  Details 
iiber  die  Darstellung  der  «eiweiBfreien  Milch »  finden  sich  ander- 
weitig. 2)  Eine  sorgfaltige  Analyse  dieses  Produktes  ergibt  fol- 
gende  Zusammensetzung : 

J)  J.  Rosenstern,  Ergebnisse  der  Inneren  Medizin  und  Kinder- 
heilkunde,  1911,  Bd.  7,  S.  385. 

2)  T.  B.  Osborne  und  L.  B.  Mendel,  Fiitterungsversuche  mit 
isolierten  Nahrungssubstanzen,  Carnegie  Institution  of  Washington,  Publi- 
cation 156,  Part  II,  1911,  S.  80—82. 


316        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Ungefahre  Zusammensetzung  der  eiweiBfreien  Milch  in 
Wasserstoff  5  Stunden  bei  100°  getrocknet: 


Unldslich  in  Wasser 


Loslich  in  Wasser 


/  Organisch         1,46  *) 
\  Anorganisch  3,93 
Lactose 


Organisch 


Anorganisch 


Atherloslich 

Stickstoff2) 

unbestimmt 


79,87 
0,13 
0,54 
2,98 


83,52 


11,09 


o/o 
5,39 


94,61 


Eine  Analyse  der  anorganischen  Bestandteile  der  protein- 
freien  Milch  zeigt,  daB  sie  enthalt: 


°/o 

Ca 

1,92 

Mg 

0,20 

Na3) 

2,18 

K 

2,82 

po4 

3,52 

CI 3) 

4,44 

so4 

0,27 

15,35 

Wie  vollstandig  ein  Zusatz  der  proteinfreien  Milch  zur 
Nahrung  anstatt  der  friiheren  kunstlichen  Salzmischung  das 


1)  Die  im  Wasser  unlosliche  Substanz  enthalt  an  Stickstoff  an- 
nahernd  0,16  °/o  der  proteinfreien  Milch  =  1,02  °/o  Eiweift.  Eine  sorg- 
faltige  Untersuchung  zeigte,  daft  die  organische  in  Wasser  unlosliche  Sub- 
stanz im  wesentlichen  aus  alien  Residuen  von  Casein  und  Lactalbumin, 
welche  nicht  niedexgeschlagen  worden  waren,  bestand. 

2)  Von  diesem  Stickstoff  sind  0,18%  durch  Tannin  niedergeschlagen, 
0,19  %  durch  Sattigung  mit  Zinksulfat  und  0,24%  durch  Phosphorwolfram- 
saure.  Damit  zeigt  sich  die  Gegenwart  von  ungefahr  1,20%  Eiweift  in  der 
wasserloslichen  Substanz.  Nach  diesen  Resultaten  erscheint  es  wahrschein- 
lich,  daft  die  eiweiftfreie  Milch  im  ganzen  2,22  %  Eiweift  enthalt,  das  macht 
0,6  %  der  an  die  Ratten  verfiitterten  Nahrung,  wenn  diese  letztere  28  % 
von  proteinfreier  Milch  enthalt  oder  3%  des  gesamten  an  die  Ratten 
verfiitterten  Proteins. 

3)  Das  Verhaltnis  von  Na  und  CI  in  der  eiweififreien  Milch  ist 
naturlich  grofter  als  in  der  Milchasche,  weil  das  Casein  durch  HC1  nieder- 
geschlagen und  das  Milchserum  durch  NaOH  neutralisiert  ist. 


Uber  Futterungsversuche  mit  isoliertcn  Nahrungssubstanzen.  317 

Problem  des  Wachstums  nach  Ftitterung  mit  einzelnen  Pro- 
teinen  gelost  hat,  wird  durch  einige  folgende  Kurven  gezeigt. 
In  diesen  vergleichenden  Versuchen,  die  teils  mit,  teils  ohne 
proteinfreie  Milch  ausgefiihrt  worden  sind,  waren  EiweiB  und 
Fett  in  bezug  auf  Quantitat  und  Qualitat  in  der  Nahrung  genau 
dieselben,  wahrend  die  Salze  und  ein  Teil  der  Kohlenhydrate 
in  den  von  Erfolg  begleiteten  Fallen  durch  eiweiBfreie  Milch 
ersetzt  worden  waren.1) 

Die  Kurven  zeigen  den  EinfluB  der  eiweiBfreien  Milch  auf 
das  Wachstum: 


Kurve  6  (Ratte  52)  und  Kurve  7  (Ratte  179  ?)  zeigen 
die  vollige  Unterdruckung  des  Wachstums  bei  verschieden 
alten  Ratten  unter  einer  Diat,  wie  sie  aus  der  Tabelle  hervor- 
geht;  Kurve  8  (Ratte  370  <?)  zeigt  das  Wachstum,  wenn  die 
Nahrung  « eiweiBfreie  Milch »  enthalt. 

*)  Zur  Erklarung  der  speziellen  Verhaltnisse  der  Nahrungsstoffe, 
wie  sie  in  den  meisten  Versuchen  dieser  Arbeit  angewandt  worden  sind, 
muB  betont  werden,  daft  diese  Verhaltnisse  aus  denjenigen  der  erfolg- 
reichen  Versuche  mit  Milchnahrung  angenommen  sind.  Spater  bringen 
wir  Studien  des  minimalen  und  optimalen  Gehalts  an  Eiweift,  Salzen  usw., 
welcher  zu  einer  entsprechenden  Ernahrung  und  einem  entsprechenden 
Wachstum  der  Ratten  notig  ist. 


318        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Nahrung : 


Ratte  52 

Ratte  179 

Ratte  370,  Periode  2 

°/o 

°/o 

°/o 

Casein  (Kuh) 

18,0 

18,0 

18,0 

«EiweiMreie  Milch» 

0,0 

0,0 

28,0 

Starke 

29,5 

32,5 

21,0—27,0 

Zucker 

15,0 

20,0—17,0 

0,0 

Agar 

5,0 

5,0 

5,0—0,0 

Salzmischung  I 

2,5 

2,5 

0,0 

Fett 

30,0 

22,0—25,0 

28,0—27,0 

In  alien  Kurven,  in  welchen  die  Nahrung  nicht  angegeben  ist,  wie 
in  der  ersten  Periode  der  Kurve  8,  war  die  Ratte  mit  gewohnlicher  ge- 
mischter  Kost  oder  clurch  die  Mutter  genahrt  worden. 


220 


Kurve  8. 


Uber  Fiitterungsversuche  mit  isolierten  Nahrungssubstanzen.  319 


-«■ 

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/ 

/ 

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/ 

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1 

e/ss  -fre/6 

Af//c/>  ~ 

(r 

¥0 

Kurve  9. 


60 


80 


Kurve  9  (Ratte  59)  und  Kurve 
10  (Ratte  193  ?)  zeigen  eine  Un- 
terdriickung  des  Wachstums 
in  verschiedenen  Altern  bei  einer 
in  der  beigegebenen  Tabelle  ge- 
zeigten  Ernahrung;  Kurve  11  (Ratte 
380?)  zeigt  das  Wachstum,  wenn 
die  Nahrung  «eiweififreie  Milch* 
enthalt. 


160 


1*0 


Edetfw  ohn  E/wvss-free Mt/ch 


^^7 


Edestin  (Hanfsamen) 

«Eiweififreie  Milch* 

Starke 

Zucker 

Agar 

Salzmischung  I 
Fett 


7         tOO         f20  fVO 

Tape 
Kurve  10. 

Nahrung : 
Ratte  59  und  193  «) 

°/o 
18,0 

0,0 
29,5 
15,0 

5,0 

2,5 
30,0 


220  2^0 


Ratte  380,  Periode 
•/. 
18,0 
28,2—28,0 
20,8—26,0 

0,0 
5,0—0,0 
0,0 
28,0 


*)  In  Periode  2  wurden  kleine  Retrage  von  «normalen»  Faeces 
gelegentlich  verfuttert,  siehe  Publication  156,  Part  II,  Carnegie  Institution 
of  Washington,  p.  61. 


320        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Kurve  12. 


Kurve  13. 


Kurve  16. 


322        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 

Kurve  12  (Ratte  56)  zeigt  ungeniigendes  Wachstum; 
Kurve  13  (Ratte  565  ?)  und  Kurve  14  (Ratte  531  zeigt 
geniigendes  Wachstum,  wenn  die  Nahrung  « eiweiBfreie 
Milch »  enthalt. 

Nahrung : 

Ratte  56    Ratte  565,  Periode  2    Ratte  531,  Periode  2 


o/o 

o/o 

°/o 

Casein  (Kuh) 

12,0 

18,0 

0,0 

Excelsin  (Brasilnufi) 

6,0 

0,0 

18,0 

«Eiweififreie  Milch» 

0,0 

28,0 

28,0 

Starke 

29,5 

28,0 

28,0 

Zucker 

15,0 

0,0 

0,0 

Agar 

5,0 

0,0 

0,0 

Salzmischung  I 

2,5 

0,0 

0,0 

Fett 

30,0 

26,0 

26,0 

Kurve  15  (Ratte  101  d")  zeigt  vollige  Unterdruckung 
des  Wachstums,  und  Kurve  16  (Ratte  284  <?)  zeigt  ein  an- 
gemessenes  Wachstum  bei  Darreichung  desselben  EiweiBkorpers, 
wenn  die  Nahrung  dabei  eiweiBfreie  Milch  enthalt. 

Nahrung : 


Ratte  101 

Ratte  284 

7° 

°/o 

Glutenin  (Weizen) 

18,0 

18,0 

«Eiweififreie  Milch* 

0,0 

28,2 

Starke 

14,5-34,5 

23,8—18,8 

Zucker 

15,0-20,0 

0,0 

Agar 

5,0 

5,0 

Salzmischung  I 

2,5 

0,0 

Fett 

45,0-20,0 

25,0-30,0 

Es  darf  in  den  vorhergehenden  Experimenten  nicht  uber- 
sehen  werden,  daB  die  « eiweiBfreie  Milch »  nicht  absolut  eiweiB- 
frei  ist.  Nach  giinstigster  Schatzung  betragt  dieses  EiweiB  aber 
hochstens  0,6  °/o  der  Nahrung.  Zunachst  ist  man  zu  glauben 
geneigt,  die  Wirksamkeit  der  eiweiBfreien  Milch  dem  Vor- 
handensein  dieses  kleinen,  kaum  schatzbaren  Retrages  an  Milch- 
eiweiB  zuschreiben  zu  miissen.  DaB  eine  solche  Erklarung  aber 
nicht  haltbar  ist,  erhellt  aus  weiteren  Versuchen,  in  welchen 
die  eiweiBfreie  Milch  unfahig  war,  das  Wachstum  zu  unterhalten, 


Uber  Fiitterungsversuche  mil  isoliertcn  Nahrungssubstanzen.  323 


wenn  sie  verschiedenen  Proteinen,  wie  Zein,  Gliadin,  Gelatin,  zu- 
gesetzt  wurde.  Mit  anderen  Worten :  die  Wirksamkeit  des  Proteins 
unserer  Nahrung  ist  mehr  dem  Gharakter  des  Hauptproteins  zu  ver- 
danken  als  irgend  einer  dem  Milchprotein  besonders  eigentiim- 
lichen  giinstigen  Beeinflussung  des  Wachstums.  Weiterhin  wird 
das  bewiesen  durch  neuere  erfolgreiche  Versuche,  in  welchen 
dieeiweiBfreie  Milch  vollstandig  durch  rein  kunstliche  Mischungen 
ersetzt  und  ihre  anorganische  Zusammensetzung  sowie  ihr  Ge- 
halt  an  Milchzucker  nachgeahmt  wurde  (siehe  Seite  351). 

Eiweifi  und  Wachstum. 

DaB  das  Wachstum  durch  eine  Mischung  von  einzelnen 
Substanzen,  in  welchen  der  N-Komponent  des  Futters  durch 
einen  einzelnen  EiweiBkorper  dargestellt  wird,  veranlaBt  werden 
kann,  zeigt  sich  deutlich  in  einigen  der  vorhergehenden  Dar- 
stellungen.  sowie  in  den  folgenden  Kurven. 

Diese  Kurven  beweisen  ein  ausreichendes  Wachstum  bei 
Verwendung  eines  einzelnen  EiweiBkorpers  im  Futter. 


zoo 


/ 

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✓ 

/ 

/  _ 
t  / 

/ 

/ 

/ 

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/ 

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 4.  

1 

1 
1 

F 

-> 

I 

80 


60 


0         20  W         60         SO         fOO        120        «H)      </60        S30  200 

Kurve  17. 

Hoppe-Seyler's  Zeitschrift  f.  physiol.  Chemie.  LXXX.  22 


Kurve  19. 


Kurve  20. 


Uber  Futterungsversuche  mit  isolierten  Nahrungssubstanzen. 


326 


Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Kurve  17  (Ratte  329  </). 
Nahrung : 

°/o 

Lactalbumin  (Kuh)  18,0 
«Eiweiftfreie  Milch»  28,2—28,0 
Starke  23,8—29,0 
Agar  5,0—  0,0 

Fett  25,0 

Kurve  18  (Ratte  258  ?). 
Nahrung: 

o/o 

Ovalbumin  (Huhn)  18,0 

«Eiweififreie  Milch*  28,2 

Starke  23,8 

Agar  5,0 

Fett  25,0 

Kurve  19  (Ratte  578  $). 
Nahrung: 


Ovovitellin  (Huhn)  18,0 

«Eiweififreie  Milch »  28,0 

Starke  28,0 

Fett  26,0 

Kurve  20  (Ratte  257  ?). 
Nahrung : 

°/o 

Glycinin  (Sojabohne)  18,0 

«Eiweififreie  Milch*  28,2 

Starke  23,8 

Agar  5,0 

Fett  25,0 
Kurve  21  (Ratte  528 
Nahrung: 

Kiirbissamen-Globulin  18,0 

«Eiweiftfreie  Milch  *  28,0 

Starke  24,0 

Fett  30,0 

Kurve  22  (Ratte  569  ?). 
Nahrung : 

Baumwollsamen-Globulin  18,0 

«Eiweiftfreie  Milch*  28,0 

Starke  28,0 

Fett  26,0 


Uber  Futterimgsversuche  mit  isolicrten  Nahrungssubstanzen.  327 


Kurve  23  (Ratte  567  J). 
Nahrung : 


Mais-Glutelin 
«EiweiMreie  Milch» 
Starke 
Fett 


o/o 
18,0 
28,0 
28,0 
26,0 


Kurve  24  (Ratte  576  ?). 
Nahrung : 


Hanfsamen-Glutelin 
«EiweiMreie  Milch » 
Starke 
Fett 


o/o 
18,0 
28,0 
28,0 
26,0 


Nicht  alle  Eiweiflkorper  fordern  das  Wachstum; 
einige  dienen  nur  der  Erhaltung  desselben,  andere  erscheinen 
sogar  zu  diesem  Zwecke  ungeeignet.  Die  folgenden  Kurven,  aus 
vielen  vergleichenden  Versuchen  gesammelt,  mogen  dies  ver- 
anschaulichen. 

Die  Kurven  zeigen  das  ungeniigende  Wachstum  bezw.  die 
Gewichtsabnahme  bei  Verabreichung  bestimmter  einzelner  Ei- 
weiBkorper  in  der  Nahrung. 


Kurve  25  (Ratte  249  $). 
Nahrung : 


Gliadin  (Weizen) 

«EiweiMreie  Milch» 

Starke 

Agar 

Fett 


o/o 
18,0 
28,2 
20,8 
5,0 
28,0 


Kurve  26  (Ratte  255  ?). 


Nahrung : 

o/o 


Hordein  (Gerste)  18,0 
«EiweiGfreie  Milch»  28,2 


Starke  18,8-12,8 


Agar  5,0 


Fett  30,0—36,0 


Uber  Fiitterungsversuche  mit  isolierten  Nahrungssubslanzen.  329 


* 

y 

s 

s 

y 

y 

s 

s 

y 

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20        W  60 


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100        120       f+O        160       180       200        220  2V0 

Tape 
Kurve  26. 

m 


120 


100 


60 


20 


20        44        60  €0 
Kurve  27. 


/ 

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L.  Ze/n. 

 -Hr 

I" 

2/ 

20        W  60 
Kurve  28. 


400  120 


Kurve  27  (Ratte  549  #). 
Nahrung : 

Roggen-Gliadin 
«Eiweififreie  Milch » 
Starke 
Fett 


> 
18,0 
28,0 
28,0 
26,0 


330        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Periode  2. 
Zein  (Mais) 
«Eiweiftfreie  Miich» 
Starke 
Agar 
Fett 
Wasser 


Kurve  28  (Ratte  503 
Nahrung: 

g 


18,0 
28,2—28,0 
23,8—24,0 
5,0—  0,0 
25,0—30,0 


Periode  3.  > 
Zein  Futter  (wie  in  Periode  2)  50,0 

Edestin  Futter  50,0 

Edestin  (Hanfsamen)  18,0 

«Eiweiftfreie  Milch »  28,0 

Starke  26,0 

Fett  28,0 


In  Periode  3  veranlaftte  der  Zusatz  von  Edestin  zur  Nahrung  auf 
einmal  eine  Wiederaufnahme  des  Wachstums.  Das  zeigt  an,  daft  Zein 
an  sich  nicht  giftig  ist. 


1/77  _^ 

—  + 

20 
Tage 

Kurve  29. 


Kurve  29  (Ratte  554  </). 
Nahrung : 

Leim  (Horn) 
«Eiweiftfreie  Milch* 
Starke 
Fett 


18,0 
28,0 
27,0 
27,0 


Ein  Studium  der  vorangegangenen  Kurven  zeigt  auf  den 
ersten  Blick,  daB  zwar  ein  geniigendes  Wachstum  bei  Ver- 
fiitterung  der  verschiedensten  Proteine  als  Quelle  des  N  moglich 
ist,  daB  ein  hinlangliches  Wachstum  aber  nicht  beobachtet 
wurde  bei  Verfutterung  eines  der  verschiedenen  Proteine,  welche, 
vom  chemischen  Standpunkt  betrachtet,  unvollstandig  sind.  Es 
ware  verfruht,  in  unseren  Fortschritten  iiber  die  strukturelle 
Zusammensetzung  der  EiweiBkorper  eine  auch  nur  annahernde 
Vorstellung  fiber  die  chemische  Anordnung  innerhalb  dieser 
komplexen  Bildungen  zu  fordern;  aber  wir  wissen,  daB  manchen 
unter  den  im  vorhergehenden  besprochenen  EiweiBkorpern  eine 
oder  mehrere  der  Aminosauren,  welche  das  gemeinsame  Kon- 
stituens  der  Albuminkomponente  sind,  fehlen.  So  dem  Weizen- 
und  Roggengliadin ;  ferner  dem  Hordein  der  Gerste  fehlen  Gly- 
kokoll1)  und  Lysin,  wahrend  das  Zein  frei  von  Tryptophan2) 


*)  Die  kleine  Quantitat  von  Glykokoll,  das  bei  einigen  Darstellungen 
von  Gliadin  erhalten  wird,  riihrt  wahrscheinlich  von  einer  geringen  Ver- 
unreinigung  aus  der  Darstellung  mit  anderen  Proteinen  her. 

2)  Fiir  die  Eigenschaften  der  pflanzlichen  Eiweiftkorper,  die  zum 


Ober  Fiitterungsversuche  mit  isolierlen  Nahrungssubslanzcn.  331 

ist.  Von  dem  Leim  mit  seinem  Mangel  an  Tyrosin,  Tryptophan 
und  Cystin  ist  es  schon  lange  bekannt,  daB  er  als  alleinige 
Quelle  des  Nahrungsstickstoffs  der  Tiere  unbrauchbar  ist.  Es 
ware  jedoch  ungerechtfertigt,  die  nutritive  Unzulanglichkeit 
eines  Proteins  dem  Fehlen  einer  einzelnen  Aminosaure  zuzu- 
schreiben.  Fur  das  Casein,  das  kein  Glykokoll  enthalt,  ist  es 
erwiesen,  daB  es  eine  der  besten  N-Quellen  fur  das  tierische 
Wachstum  ist. 

Warum  wachsen  die  Tiere  bei  gewissen  Ernahrungsformen  nicht? 

Man  mochte  bei  weniger  ernstem  Uberlegen  geneigt  sein, 
das  Ausbleiben  des  Wachstums  bei  einigen  der  im  vorher- 
gehenden  berichteten  Ernahrungsarten  dem  Mangel  von  ge- 
niigender  Nahrungszufuhr  zuzuschreiben,  i.  e.  der  Unzulanglich- 
keit vom  Standpunkt  des  Energiegleichgewichts.  DaB  diese 
Erklarung  nicht  geniigen  kann,  erhellt  aus  einer  Studie  iiber 
den  tatsachlichen  Nahrungsverbrauch.1)  Da  die  kalorischen 
Werte  dieser  Fiitterungen  annahernd  die  gleichen  waren,2) 
namlich  ca.  5,2  Kalorien  per  Gramm,  war  die  den  Ratten  ge- 
lieferte  Energie  ihrem  Nahrungsverbrauch  genau  proportional, 
ausgenommen  wenn  Zein  verfuttert  wurde.  Der  kalorische 
Wert  dieser  letzteren  Ernahrung  betrug  ca.  4,5  Kalorien  per 
Gramm,  weil  es  notig  war,  das  Zein  zum  Zwecke  einer  guten 
Ausnutzung  zu  wassern,  bevor  man  es  mit  den  andern  Bestand- 
teilen  mischte.  Die  Kurven  30  und  31  liefern  eine  Illustration 
zu  der  Unhaltbarkeit  obiger  Annahme,  welche  die  Wachstums- 

besonderen  Studium  ausgewahlt  wurden,  und  zwar  wegen  der  Leichtig- 
keit,  mit  welcher  sie  der  notwendigen  Reinigung  unterworfen  werden 
konnen,  siehe  T.B.Osborne,  Ergebnisse  der  Physiologie,  1910,  Bd.  10, 
S.  47. 

1)  Die  Darreichung  des  Futters  in  Form  einer  Paste  von  solcher 
Konsistenz,  daft  die  Ratten  es  nicht  zerstreuen  konnen,  bat  es  moglich 
gemacht,  ziemlich  genaue  Zahlen  iiber  die  verzehrten  Mengen  zu  erhalten. 
Die  wochentliche  Nahrungszufuhr  ist  auf  den  meisten  unserer  Tabellen 
verzeichnet. 

2)  Neue  Bestimmungen  iiber  die  meisten  der  in  vorliegenden  Ver- 
suchen  angewandten  Proteine  siehe  F.  G.  Benedict  und  T.  B.  Osborne, 
Journal  of  Biological  Chemistry,  1907,  Bd.  3,  S.  119—133. 


332        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


hemmung  im  Futtermangel  suchen  will.  Hier  sehen  wir  die 
Aufzeichnungen  iiber  junge  Tiere  in  vergleichbaren  Altern  und 
auf  fast  gleicher  Entwicklungsstufe,  welche  eine  fast  gleiche 
Nahrungszufuhr  erhalten  haben.3)  Die  mit  Gliadin  gefiitterten 
sind  verkummert,  wahrend  die  mit  Casein  gefiitterten  Ratten 
ein  normales  Wachstum  zeigen. 


Kurve  30. 


Kurve  30  (Ratte  254  ?)  Gliadinfutterung,  Kurve  31  (Ratte 
566  d")  Gaseinfiitterung.  Ein  Vergleich  dieser  Kurven  zeigt  die 
ahnliche  wochentliche  Nahrungszufuhr  der  2  Tiere.  Der  wesent- 
liche  Unterschied  in  der  Ernahrung  liegt  im  Charakter  der  Ei- 
weiBkorper.  Dieser  letztere  muB  fiir  die  Differenz  im  Wachstum 
in  den  2  sonst  durchaus  vergleichbaren  Versuchen  verantwort- 
lich  gemacht  werden. 


3)  Dieser  Punkt  mufi  im  Auge  behalten  werden,  seit  es  anerkannt 
ist,  dafi  der  Energiebedarf  (und  folglich  die  Nahrungszufuhr)  mit  dem 
Grofierwerden  des  Tieres  wachsen  mufi.  Die  Vergleiche  mussen  also 
auf  Grund  der  Grofie  und  nicht  des  Alters  vorgenommen  werden. 


Uber  Fiitterungsversuche  mit  isolierten  Nahrungssubstanzen.  333 


Nah rung : 


Ratte  254 

Ratte  566 

o/o 

«/0 

Gliadin  (Weizen) 

18,0 

0,0 

Casein  (Kuh) 

0,0 

18,0 

Eiweififreie  Milch 

28,2 

28,0 

Starke 

20,8 

28,0 

Agar 

5,0 

0,0 

Fett 

28,0 

26,0 

20 


¥0 

rage 
Kurve  31. 


Fur  das  ungleiche  Wachstum 
bei  verschiedener,  allerdings  nur  im 
Hinblick  auf  die  EiweiBkorper 
wechselnder  Ernahrung  kommt  in 
Betracht:  eine  ungleiche  Verdau- 
lichkeit  und  Ausnutzbarkeit  der 
verschiedenen  EiweiBkorper  bezw. 
Nahrungsgemische.  Die  Moglichkeit 
eines  geringeren  Nahrwertes  jener 
Nahrung,  die  sich  fur  das  Wachs- 
tum als  minderwertiger  erwiesen 
hat,  kann  nicht  vollstandig  aus- 
geschlossen  werden  ohne  hinrei- 
chende  Bilanzversuche.  Mendel 
und  Fine1)  haben  in  der  Tat  einige 
Differenzen  zwischen  den  verfut- 
terten  Proteinen  durch  Versuche 
an  Menschen  und  Hunden  entdeckt.  Weizen-  und  Gerste- 
produkte  wurden  aber  regelmaBig  als  gut  ausnutzbar  be- 
funden;  und  es  ist  sicher,  daB  diese  Gruppe,  der  das  Gliadin, 
Hordein  und  Glutenin  angehort,  in  auffallend  ungleicher  Weise 
auf  die  Wachstumserscheinungen  der  Ratten  einwirkt.  Bedacht 
muB  werden,  daB  die  EiweiBkorper  vor  der  Futterung  vollig 
isoliert  wurden,  sodaB  die  Stoning  der  unverdaulichen  Bei- 
mischungen  in  Form  der  Cellulose  und  Hemicellulose  bezw. 
anderer  der  Verdauung  hinderlicher  Zellenbestandteile  aus  der 
Betrachtung  ausgeschaltet  ist.  Die  Tatsache,  daB  wir  mehrere 


/r— — — 

?  

s 

1 

/ 

/ 

/ 

/ 

1 

t 

1 

-t  

1 

/ 

/  / 
/  / 

I  / 

 -> 

so 


l)  L.  B.  Mendel  and  M.  S.  Fine,  Journal  of  Biological  Chemistry, 
1911,  Bd.  10,  S.  303,  339,  345,  433;  1912,  Bd.  11,  S.  1  und  5. 


334         Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 

Ratten  iiber  Perioden  von  500  Tagen  bei  einer  Nahrung,  in 
der  als  alleiniges  Protein  das  Gliadin  gereicht  wurde,  erfolg- 
reich  futtern  konnten,  ist  ein  guter  Beweis  fur  die  glanzende  Ver- 
wertbarkeit  des  Gliadins.  Beschrankte  Quantitaten  von  anderen 
entsprechenden  EiweiBkorpern  wie  Casein  oder  Edestin  fiihren 
bald  zu  einer  Herabsetzung  des  Korpergewichts.  Wir  haben 
also  keinen  Grund  zu  der  Annahme,  daB  Gliadin  nicht  ebenso 
ausnutzbar  wie  andere  EiweiBkorper  ist. 

EiweiBkorper  der  Leguminosen  und  Wachstum. 

Wir  wollen  nun  nach  vorhergegangenen  Gesichtspunkten 
die  EiweiBkorper  der  Leguminosen  betrachten.  Trotz  der  Tat- 
sache,  daB  die  modernen  Methoden  der  Proteinhydrolyse  und  die 
Bestimmung  der  Aminosaure  diese  EiweiBkorper  als  vollstandig 
bezeichnen,  gelang  es  uns  doch  nicht,  mit  einer  Ernahrung,  in 
welcher  die  Leguminosenproteine  die  einzige  N-Quelle  waren, 
Wachstum  zu  erzielen,  ausgenommen  mit  den  EiweiBkorpern  der 
Sojabohne.  Dies  erhellt  deutlich  aus  den  beigegebenen  Kurven 
(32  und  33),  in  welchen  die  Verwendung  des  Phaseolins  *)  der 
Schminkbohne,  Phaseolus  vulgaris,  als  einzig  verfutterter  EiweiB- 
korper sofort  von  Wachstumstillstand  und  Gewichtsabfall  be- 
gleitet  ist,  wahrend  schnell  wieder  ein  Ansatz  und  eine  Gewichts- 
zunahme  erfolgt,  wenn  dieses  Phaseolin  durch  Milchcasein  oder 
Edestin  des  Hanfsamens  ersetzt  wird.  Diese  Wachstumshemmung 
ist  nicht  nur  ein  Gharakteristikum  der  ersten  Entwicklungs- 
periode,  sie  erfolgt  vielmehr  in  verschiedenen  Jugendstadien. 
Und  sie  wird  in  ahnlicher  Weise  illustriert  in  den  Versuchs- 
tabellen  mit  Konglutin  aus  den  gelben  Lupinen,  Lupinus  luteus, 
Kurve  34;  Erbsenlegumin  aus  der  Gartenerbse,  Pisum  sativum, 
Kurve  35;  Vignin  aus  der  Kuherbse,  Vigna  catjang,  Kurve  36; 
Legumelin  aus  der  Sojabohne,  Soja  hispida,  Kurve  37. 

Die  nachfolgenden  Kurven  zeigen  den  EinfluB  der  Legu- 
minoseneiweiBkorper  auf  das  Wachstum. 

')  In  bezug  auf  Herstellung  dieses  Praparats  vgl.  T.  B.  Osborne, 
Abderhaldens  Handbuch  der  biochemischen  Arbeitsmethoden,  1909, 
Bd.  2,  S.  311. 


Uber  Futterungsversuche  mit  isolierlen  Nahrungssubgtanzen. 


335 


Kurve  33. 


Kurve  34. 


Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


120 


y 

/ 

/ 

✓ 

/ 

 A- 

/ 

/ 

/ 

/ 

/ 

/ 



/ 

f 

<— - 

^  «tf  <fc? 

Kurve  37. 


Kurve  32  (Ratte  389  ?) 
Phaseolinfutterung.  —  Kurve 
33  (Ratte  498  ?)  Phaseolin- 
fiitterung.  Diese  Tabellen 
zeigen  die  vollige  Unterdrtik- 
kung  des  Wachstums,  wenn 
das  Phaseolin  der  Schmink- 
bohne  den  einzigen  Eiweifi- 
korper  der  Nahrung  bildet. 
Die  Wiederaufnahme  des 
Wachstums  erfolgte  in  da- 
rauffolgenden  Perioden  (3,  5, 
7,  9),  wenn,  wie  die  Futte- 
rungstabelle  zeigt,  ein  ande- 
rer  EiweiBkorper  an  dessen 
Stelle  gesetzt  wurde. 


Uber  Fiitterungsversuche  mit  isoliertcn  Nahrungssubstanzen.  337 


Nali  rung : 


Ratte  389 

l<  OCJ«7 

i  tan  i  tjiju 

Periode  2,  4,  6,  8 

rcilOUvJ  0,0,  / ,  1/ 

17  CI1UU.C  ±\J 

Ratte  498 

Ratte  498 

Ratte  498 

Periode  2  und  4 

Periode  3 

Periode  5 

°/o 

0  / 

7° 

0  / 

Phaseolin  (Schminkbohne) 

18,0 

U,U 

n  c\ 
U,U 

Casein  (Kuh) 

0,0 

18,0 

0,0 

Edestin  (Hanfsamen) 

0,0 

0,0 

18,0 

«Eiweiftfreie  Milch » 

28,2 

28,2-28,0 

0,0 

«Kiinstlich  eiweiftfr.  Milch » 

0,0 

0,0 

29,5 

Starke 

23,8 

23,8-27,0 

24,5 

Agar 

2,0 

5,0—  0,0 

0,0 

Fett 

28,0 

25,0—27,0 

28,0 

Kurve  34  (Ratte  575  ¥ )  Futterung  mit  Konglutin.  Kurve  35 
(Ratte  563  <?)  Futterung  mit  Erbsenlegumin.  Kurve  36  (Ratte 
518  ?)  Futterung  mit  Vignin.  Kurve  37  (Ratte  527  c?)  Futterung 
mit  Legumelin. 


Nahrung: 

Ratte  575 

Ratte  563 

Ratte  518 

Ratte  i 

°/o 

o/o 

o/o 

o/o 

Konglutin  (gelbe  Lupinen) 

18,0 

0,0 

0,0 

0,0 

Legumin  (Gartenerbse) 

0,0 

18,0 

0,0 

0,0 

Vignin  (Kuherbse) 

0,0 

0,0 

18,0 

0,0 

Legumelin  (Sojabohne) 

0,0 

0,0 

0,0 

18,0 

«Eiweififreie  Milch* 

28,0 

28,0 

28,0 

28,0 

Starke 

28,0 

28,0 

28,0 

22,0 

Fett 

26,0 

26,0 

26,0 

32,0 

Ein  Vergleich  der  taglichen,  Phaseolin  enthaltenden  Nah- 
rung mit  der  taglichen,  Casein  oder  Edestin  enthaltenden  zeigt, 
daft  fur  den  Gewichtsverlust  wahrend  der  Perioden,  in  welchen 
Phaseolin  gereicht  wurde,  nicht  eine  ungeniigende  Nahrungs- 
zufuhr verantwortlich  gemacht  werden  kann.  Wenn  die  Nahrungs- 
zufuhr  namlich  durch  das  Verhaltnis  von  Gramm  Nahrung  zu 
Gramm  Ratte  ausgedruckt  wird,  so  findet  man,  daB  wahrend 
der  verschiedenen  Futterungsperioden  mit  verschiedenen  Pro- 
teinen  in  dieser  Beziehung  nur  eine  geringe  Differenz  besteht. 
Dabei  muB  besonders  erwogen  werden,  daB  nach  Erreichung 
eines  Gewichts  von  ca.  70  g  die  normale  mittlere,  taglich  ein- 


338        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


genommene  Nahrung  sich  gewohnlich  per  Gramm  Korper- 
gewicht  zu  vermindern  beginnt,  wenn  die  Ratte  mit  ahnlichen, 
entsprechende  Proteine  enthaltenden  Mischungen  weiter  ge- 
futtert  wird.  Ferner  waren  in  zahlreichen  Versuchen  mit  Gliadin 
oder  Hordein  ahnliche  Quantitaten  von  Nahrung  vollstandig 
geniigend,  urn  die  Ratten  iiber  langere  Perioden  auf  ent- 
sprechender  GroBe  zu  erhalten.  Dagegen  haben  wir  keine 
Kenntnis  von  der  Ausdehnung,  bis  zu  welcher  das  Phaseolin 
verwertet  wird.  Daten  fur  einen  Vergleich  sind  im  folgenden 
zusammengestellt. 


Zusammenstellung  iiber  Nahrungszufuhr  bei  mit 
Phaseolin  gefutterten  Ratten. 

Ratte  389  (Kurve  32). 


Periode 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Nahrung 

Pha- 
seolin 

Ca- 
sein 

Pha- 
seolin 

Ca- 
sein 

Pha- 
seolin 

Ca- 
sein 

Pha- 
seolin 

Ca- 
sein 

Ede- 
stin 

Mittleres  Gewicht 
der  Ratte 

g 
48,4 

g 

57,2 

g 
67,4 

g 
77,2 

g 
84,5 

g 
92,1 

g 

100,8 

g 

105,6 

g 

130,2 

Durchschnittl.  tag- 
liche  Nahrungszufuhr 

4,0 

4,7 

6,15 

8,3 

6,26 

8,74 

6,87 

7,6 

7,4 

Nahrung  per  Tag 
und  Gramm  Ratte 

0,082 

0,082 

0,091 

0,107 

0,074 

0,095 

0,068 

0,072 

0,057 

Ratte  498  (Kurve  33). 


Periode 

2 

3 

4 

5 

Nahrung 

Phaseolin 

Casein 

Phaseolin 

Edestin 

Mittleres  Gewicht  der  Ratte 

g 
30,8 

g 
44,0 

g 
54,2 

g 
69,7 

Durchschnittliche  tagliche 
Nahrungszufuhr 

3,5 

5,4 

4,7 

5,2 

Nahrung  per  Tag  und  Gramm 
Ratte 

0,113 

0,122 

0,086 

0,075 

Uber  Fiitterungsversuche  mit  isoliertcn  Nahrungssubstanzen.  339 


Wir  wollen  noch  kein  abschlieBendes  Urteil  dariiber  ab- 
geben,  ob  die  ungunstigen  Resultate  mit  den  Leguminosenver- 
suchen  der  geringeren  Ausnutzbarkeit  allein  zugeschrieben 
werden  sollen,  trotz  ungunstiger  Resultate,  die  in  dieser  Hin- 
sicht  bei  anderen  Tieren  erhalten  worden  sind.1)  Vielmehr 
verlangt  die  Frage,  warum  die  Ratten  die  Legurainosennahrung 
nicht  mit  solchem  Appetit  verzehren,  wie  ein  Futter,  das  aus 
anderen  Pflanzen  isolierte  Proteine  enthalt,  noch  nahereErklarung. 
Es  ist  tatsachlich  ja  sehr  leicht  zu  begreifen,  daB  die  Legu- 
minosendiat  Ernahrungsstorungen  verursacht  und  dadurch  Ge- 
legenheit  zu  Gewichtsstiirzen  gibt.  Es  ist  aber  anderseits  schwer, 
wenn  nicht  gar  unmoglich,  in  dieser  Beziehung  zwischen  Ur- 
sache  und  Wirkung  zu  unterscheiden.  Wenn  der  Wachstums- 
stillstand  einer  Unfahigkeit  des  Proteins  zur  Unterhaltung  des 
Wachstums  zuzuschreiben  ist,  dann  ist  es  leicht  begreiflich,  daB 
Appetit  des  Tieres  wie  Nahrungsbedarf  auf  eine  Unterhaltungs- 
stufe  zunickgedrangt  werden  konnen;  anderseits  ist  es  ebenso 
klar,  daB,  solange  die  Nahrungszufuhr  fur  nicht  mehr  als  die 
Unterhaltung  genugt,  ein  wirkliches  Wachstum  nicht  erfolgen 
kann.  Es  ist  eine  auffallende  Tatsache,  daB  in  alien  unseren 
zahlreichen  Versuchen,  in  welchen  bei  den  Tieren  Wachstums- 
stillstand  bestand,  der  wochentliche  Nahrungsverbrauch  bei 
Ratten  derselben  GroBe  nahezu  derselbe  war,  ganz  gleich  welches 
Protein  immer  auch  angewandt  wurde. 

Ein  Punkt  verlangt  in  unseren  Versuchen  spezielle  Be- 
achtung.  Die  Resultate  konnen  nicht  zufalligen  und  auBeren 
Gninden  zugeschrieben  werden,  denn  die  Versuche  sind  ofters 
gemacht  worden,  und  zwar  in  einer  Ausdehnung,  die  hier  nicht 
naher  erortert  werden  kann.  Man  ist  zu  dem  Schlusse  geneigt, 
daB  die  Leguminosen,  im  Gegensatz  zu  den  Cerealien,  nicht 
als  der  HaupteiweiBfaktor  in  der  Diat  fur  Mensch  und  Tier 
gelten  konnen.  Es  sind  aber  noch  mehr  Versuche  erforder- 
lich,  ehe  eine  solche  Theorie  als  feststehend  angenommen 
werden  darf. 


cf.  L.  B.  Mendel  and  M.  S.  Fine,  Journal  of  biological  che- 
mistry, 1912,  Bd.  10,  S.  433. 

Hoppe-Seyler's  Zeitschrift  f.  physiol.  Chemie.    LXXX.  23 


340        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Quantitative  Gesichtspunkte  iiber  Wachstumshemmung. 

Soweit  haben  wir  tins  rait  den  qualitativen  Unterschieden 
der  Nahrungssubstanzen,  welche  mit  dem  Erfolg  oder  MiBerfolg 
bei  unseren  Fiitterungsversuchen  an  jungen  Tieren  in  Zusammen- 
hang  stehen  mogen,  beschaftigt.  Es  folgt  aber  daraus  keines- 
falls,  daB  die  bisher  angewandten,  ziemlich  gleichartigen  Fiitte- 
rungsbedingungen  —  Bedingungen,  die  zum  Teil  durch  die  Frage- 
stellung  der  Arbeit  gegeben  sind  —  das  Optimum  im  Hinblick 
auf  die  Quantitat  der  angewandten  Nahrstoffe  bilden.  Uber- 
triebene  Ansichten  iiber  die  Wichtigkeit  einer  Zufuhr  hoher 
EiweiBmengen  fur  das  Wachstum  sind  noch  weit  verbreitet.  Die 
von  uns  eingefuhrte  Futterungsmethode  weist  befriedigende  Wege 
zur  Priifung  der  notwendigen  EiweiBmengen  unter  kontrollier- 
baren  Bedingungen.  Wir  verglichen  die  Wachstumsforderung 
durch  Nahrungsreichungen,  die  ungleiche  Mengen  von  Protein 
enthielten,  miteinander.  Bei  diesen  angewandten  Mengen  wurde 
das  EiweiB  durch  Kohlenhydrate  (oder  umgekehrt)  vom  Stand- 
punkte  der  Energievalenz  in  wesentlich  isodynamen  Betragen 
ersetzt.  Die  Nahrungsgemische,  berechnet  fur  ca.  5  Kalorien 
per  Gramm,  andern  in  ihrem  EiweiBgehalt  wie  folgt: 


Eiweift 


Casein 


Edestin 


o/o 

o/o 

o/o 

o/o 

o/o 

o/o 

o/o 

o/o 

o/o 

o/o 

o/o 

4,0 

6,5 

9,0 

12,0 

18,0 

31,0 

4,0 

6,5 

9,0 

12,0 

18,0 

31,0 

0,76 

1,12 

1,44 

1,83 

2,65 

4,38 

0,92 

1,29 

1,74 

2,19 

3,24 

5,48 

3  + 

5 

7  + 

9,6 

14,4 

25 

3  + 

5 

7  + 

9,6 

14,4 

25 

38 

39 

40 

41 

42 

43 

44 

45 

46 

47 

48 

49 

Gehalt  an 
Eiweift 

Gehalt  an  N 

Eiweift- 
kalorien 

Vgl.  Kurve  Nr. 


Den  Tieren  wurden  unbegrenzte  Mengen  der  betreffenden 
Nahrung  gegeben  und  der  jeweilige  Verbrauch  gemessen.  Einige 
der  Resultate  sind  in  den  folgenden  Tabellen  wiedergegeben. 
Die  Kurven  zeigen  den  EinfluB  des  EiweiBgehalts  der  Nahrung 
auf  das  Wachstum. 


Uber  Fiitterungsversuche  mit  isolierten  Nahrungssubstanzen.  341 
Versuche  mit  Casein. 


Kurve  38  (Ratte  341  ?)  zeigt  ein  Absinken  des  Gewichts 
bei  einer  Diat,  die  4°/o  Casein  enthalt. 

Nahrung : 


o/o 

Casein  (Kuh) 

4,0 

«EiweiMreie  Milch » 

28,0 

Starke 

22,0 

Lactose 

14,0 

Agar 

5,0 

Fett 

27,0 

Kurve  39  (Ratte  346  ?)  zeigt  keine  Wachstumszunahme 
bei  einer  Diat,  die  6,5°/o  Casein  enthalt. 


Nahrung : 


°/o 

Casein  (Kuh) 

6,5 

«EiweiMreie  Milch » 

28,0 

Starke 

22,0 

Lactose 

11,5 

Agar 

5.0 

Fett 

27,0 

Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Kurve  40  (Ratte  351  j)  zeigt  Unterdruckung  des  Wachs- 
tums  bei  einer  Diat,  die  9°/o  Casein  enthalt. 

Nahrung: 


Casein  (Kuh) 

«EiweiMreie  Milch; 

Starke 

Lactose 

Agar 

Fett 


> 
9,0 
28,0 
22,0 
9,0 
5,0 
27,0 


/ 

/ 

A 

—  r 
/ 

/ 

/ 

/ 

/ 

t 

1 

1 

i 

J 

Casern 

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/ 

/ 

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/ 

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ff"  

ty 

X, 

i       ..  .. 

s 

f 

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/ 

/ 

r  ' 

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20 


40         60  60 
Tape 

Kurve  41. 


f20 


60 


rape 
Kurve  42. 


Kurve  41  (Ratte  323  </)  zeigt  ungeniigendes  Wachstum 
bei  einer  Diat,  die  12°/o  Casein  enthalt. 

Nahrung: 


Casein  (Kuh) 
«EiweiMreie  Milch  i 
Starke 
Lactose 
Agar 
Fett 

Kurve  42  (Ratte  545  ?)  zeigt  normales  Wachstum  bei 
einer  Diat,  die  18°/o  Casein  enthalt.  Die  gleiche  Quantitat 
wurde  in  den  meisten  unserer  Versuche  angewandt. 


•/• 
12,0 
28,0 
22,0 
6,0 
5,0 
27,0 


liber  Fiitterungsversuchc  mit  isolierten  Nahrungssubstanzen.  343 


Nahrung: 


Casein  (Kuh) 


Starke 
Fett 


18,0 


tEiweififreie  Milch »  28,0 


28,0 
26,0 


Kurve  43  (Ratte  326  ?).  Die  Diat  ent- 
hielt  31°/o  Casein. 

Nahrung : 

°/o 

Casein  (Kuh)  31,0 

«Eiweififreie  Milch »  28,0 

Starke  9,0 

Agar  5,0 

Fett  27,0 


s 

/ 

/ 

/ 

/ 

-t  

~asein_ 

2 

i  

Tage 
Kurve  43. 


Versuche  mit  Edestin. 

Kurve  44  (Ratte  357  ?)  zeigt  ein  Abfallen  des  Gewichts 
bei  einer  Diat,  die  4°/o  Edestin  enthalt. 


Nahrung: 


Edestin  (Hanfsamen) 
«Eiweiflfreie  Milch  * 
Starke 
Lactose 
Agar 
Fett 

"  0         20         +0         60  80         WO  f20 

Tage 

Kurve  44. 


Uber  Fiitterungsversuche  mit  isolierten  Nahrungssubstanzon.  345 


Kurve  45  (Ratte  366  #)  zeigt  Gewichtsstillstand  bei  einer 
Diiit,  die  6,5°/o  Edestin  enthalt,  und  Wiederaufnahme  des 
Wachstums,  wenn  der  EiweiBgehalt  der  Nahrung  vermehrt  wird. 

Nahrung: 


Periode  2 

Periode  3 

0/0 

o/o 

Edestin  (Hanfsamen) 

6,5 

18,0 

«Eiweififreie  Milch* 

28,0 

28,0—29,5 

Starke 

20,0 

26,0—24,5 

Lactose 

11,5 

0,0 

Agar 

5,0 

0,0 

Fett 

29,0 

28,0 

Kurve  46  (Ratte  368  ?)  zeigt  eine  leichte  Gewichts- 
zunahme  bei  einer  Diat,  die  9°/o  Edestin  enthalt. 

Nahrung: 


> 

Edestin  (Hanfsamen) 

9,0 

«Eiweiftfreie  Milch » 

28,0 

Starke 

20,0 

Lactose 

9,0 

Agar 

5,0 

Fett 

29,0 

160 


1¥0 


120 


100 


80 


60 


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C-  

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y 

/ 

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/ 

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0         20        ¥0  60 


fOO       120       1*0       160  *80 


Tape 
Kurve  47. 


346        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Kurve  47  (Ratte  369  ?)  zeigt  Wachstum  bei  einer  Diat, 
welche  12°/o  Edestin  enthalt. 

Nahrung: 

Edestin  (Hanfsamen)  12,0 

«EiweiMreie  Milch  *  28,0 

Starke  20,0-25,0 

Lactose  6,0 

Agar  5,0-0,0 

Fett  29,0 


Kurve  48  (Ratte  372  <?)  zeigt  normales  Wachstum  bei 
einer  Diat,  welche  1 8  °/o  Edestin  enthalt.  Die  gieiche  Quantitat 
wurde  in  den  meisten  unserer  Versuche  angewandt. 


Uber  FUtterungsversuche  mit  isolierten  Nahrungssubstanzen.  447 


Nahrung: 

°/o 

Edestin  (Hanfsamen)  18,0 

«EiweiMreie  Milch*  28,0 

Starke  19,0-26,0 

Agar  5,0—0,0 

Fett  30,0—28,0 


Kurve  49. 


Kurve  49  (Ratte  367  </•)  zeigt,  daB  das  Wachstum  bei 
einer  Diat,  die  31°/o  Edestin  enthalt,  nicht  zunimmt,  obwohl  der 
durch  unsere  Standarddiat  festgesetzte  Betrag  iiberstiegen  wird. 

Nahrung: 


Edestin  (Hanfsamen)  31,0 

«EiweiGfreie  Milch*  28,0 

Starke  7,0 

Agar  5,0 

Fett  29,0 


348        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 

In  bezug  auf  diese  Daten  muB  hervorgehoben  werden, 
dafi,  was  den  Proteinverbrauch  betrifft,  die  untere  Wachstums- 
grenze  mit  7 — 9°/o  Protein  (6°/o  Proteinkalorien)  im  Nahrungs- 
gemisch  erreicht  wurde.  Mit  18°/o  (14— 15°/o  Proteinkalorien) 
war  ein  entsprechendes  Wachstum  gesichert,  dieses  wurde 
aber  nicht  gefordert  durch  ein  fiber  diesen  Betrag  hinaus- 
gehendes  MaB  von  Protein,1)  Ersichtlich  ist  das  Wachs- 
tum nicht  der  EiweiBzufuhr  proportional,  obwohl  ein 
gewisser  minimaler  Gehalt  an  EiweiB  durchschnittlich  ohne  Ein- 
fluB  auf  das  Wachstum  bleibt.2)  Ahnlich  gibt  es  einen  minimalen 
und  maximalen  Gehalt  der  « Mineral nahrstoffe»,  welche  die  Mog- 
lichkeit  des  Wachstums  bedingen.  Ein  Vergleich  der  Wachstums- 
kurven  von  Tieren,  welche  in  ihrer  Nahrung  verschieden  hohe 
Mengen  derselben  Salzmischung  erhalten  (eiweiBfreie  Milch), 
zeigt  die  untere  Grenze  fur  den  Bedarf  an  anorganischen  Salzen. 
Der  Charakter  der  Nahrung,  der  mit  Ausnahme  des  Gehalts 
an  anorganischen  Salzen  konstant  bleibt,  wird  im  AnschluB  an 
die  folgenden  Kurven  gezeigt: 


°/o 

°/o 

o/o 

Gehalt  an  Eiweifi 

18 

18 

18 

18 

Gehalt  an  «eiweififreier  Milch» 

7 

U 

21 

28 

Gehalt  an  mineralischen  Nahrstoffen 

1,05 

2,10 

3,15 

4,20 

Vergleiche  die  Kurven  Nr. 

50 

51 

52 

58 

Die  Kurven  zeigen  den  EinfluB  desBetrages  an  anorganischen 
Salzen  auf  das  Wachstum. 


x)  In  einem  Bericht  iiber  die  zur  Inanition  fiihrenden  Bedingungen 
schreibt  Rosenstern  (Ergebnisse  der  inneren  Medizin  und  Kinderheil- 
kunde,  1911,  Bd.  7,  S.  390):  «Eiweifihunger  spielt  im  Sauglingsalter  eine 
sehr  geringe  Rolle  und  kommt  in  Anbetracht  des  hohen  Eiweifigehalts 
der  Kuhmilch  praktisch  kaum  in  BetrachU.  Der  Betrag  an  Eiweifi  iiber- 
steigt  in  den  festen  Bestandteilen  der  menschlichen  Milch  selten  12°/o. 

2)  Wir  miissen  die  Tatsache  beachten,  dafi  in  verschiedenen  Ver- 
suchen  mit  verhaltnismafiig  hohem  Eiweifigehalt  (31°/o)  im  Nahrungs- 
gemisch  die  jungen  Ratten  in  wenigen  Tagen  zugrunde  gingen.  Weitere 
Beobachtungen  in  dieser  Beziehung  sind  wiinschenswert,  bevor  definitive 
Schlufifolgerungen  berechtigt  sind. 


Uber  Futtcrungsversuche  mit  isolierten 


Nahrungssubslanzen.  349 


Kurve  50  (Ratte  388  f)  zeigt  Wachstumszunahrae  bei 
einer  Diat,  welche  des  in  unserer  Standardnahrung  befind- 
lichen  Betrags  an  anorganischen  Salzen  enthalt. 

Nahrung: 

0/0 

Casein  (Kuh)  18,0 
«EiweiMreie  Milch »  7,0 
Starke  22,0—27,0 
Lactose  21,0 
Agar  5,0—0,0 
Fett  27,0 

Kurve  51  (Ratte  391  </)  zeigt  das  Wachstum  bei  einer 
Diat,  welche  lh  des  in  unserer  Standardnahrung  befindlichen 
Betrags  an  anorganischen  Salzen  enthalt. 


350        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


220 


200 


ISO 


t60 


120 


100 


80 


60 


20 
0 


Nahrung: 

Casein  (Kuh) 

«EiweiMreie  Milch » 

Starke 

Lactose 

Agar 

Fett 


•/• 
18,0 
14,0 
22,0-27,0 

14,0 
5,0—0,0 
27,0 


J  / 

/  / 

/ 

J / 

— 

/ 

/ 

u 

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/  / 

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1 

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frtve/ss 

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Casein 

/cfi  ft  "A 

 ^ 

21 

l/ 

20         W         60        80         WO        420  460 

Tage 
Kurve  51. 

Kurve  52  (Ratte  386  <?)  zeigt  das  Wachstum  bei  einer 
Diat,  welche  3k  des  in  unserer  Standardnahrung  befindlichen 
Betrags  an  anorganischen  Salzen  enthalt. 

Nahrung: 


°/o 

Casein  (Kuh) 

18,0 

«EiweiMreie  Milch  » 

21,0 

Starke 

22,0—27,0 

Lactose 

7,0 

Agar 

5,0—0,0 

Fett 

27,0 

Uber  Fiittcrungsversuche  mit  isolierten  Nahrungssubstanzen.  351 


Kurve  53  (Ratte  379  #)  zeigt  zum  Vergleich  das  normale 
Wachstum  bei  unserm  Standardnahrungsgemisch. 

Nahrung: 

°/o 

Casein  18,0 
«Eiweiftfreie  Milch.  28,2—28,0 
Starke  23,8—27,0 
Agar  5,0—0,0 
Fett  25,0-27,0 

Kunstliche  Salzmischungen  und  Wachstum. 

Wenn  wir  die  Tatsache,  dafi  ein  Wachstum  der  Ratten 
nur  dann  erfolgt,  wenn  in  den  verabreiehten  Mischungen  ver- 
schiedener  Nahrungssubstanzen  die  «eiweiBfreie  Milch »  einen 
Teil  der  Zutaten  ersetzt,  zu  erklaren  versuchen,  fallen  uns 


352 


Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


220 


200 


160 


<20 


80 


*0 


/ 

— r — 
/ 

/ 

/ 

/ 

/ 

/ 

/ 

i 

A 

—A  

/ 

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f 

II 

t— 

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Lt-  

/  / 

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J/ 

<  

—  f/m 

iss-firet'e 

Mm  ^ 
tern 

1%  -  - 

0         20        W        60        80        100        120       1+0       160  180 

Tagre 
Kurve  53. 


verschiedene  Moglichkeiten  auf.  Die  ersten  Salzmischungen, 
wie  die  von  uns  nach  Rohmann  und  McGollum  modifizierten, 
reprasentieren  nicht  genau  die  Zusammensetzung  der  Milch- 
salze,  wie  sie  in  ihrem  natiirlichen  Medium  vorhanden  sind. 
Es  ist  leieht  moglich  und  in  andern  Gebieten  der  biologischen 
Forschung  keineswegs  ohne  Analogon,  daB  ein  genau  bestimmtes 
Gleichgewicht  der  verschiedenen  Ionen  angenommen  werden 
muB,  um  das  physiologische  Gleichgewicht  zu  erhalten  und 
eine  geordnete  Zellaktivitat  zu  erregen,  wie  das  Wachstum 
eine  ist.  Es  ist  eben  begreiflich,  daB  eine  Umordnung  der 
verschiedenen  eigenartigen  Komplexe  von  organischen  und  an- 
organischen  Radikalen,  wie  sie  bei  dem  Veraschen  der  Milch 


Uber  Fiitterungsversuche  mil  isolierlen  Nahrungssubstanzen.  353 


vorkommt,  die  ideale  Wirksamkeit  der  anorganischen  Ionen  der 
Nahrung  aufhebt.  Diese  Moglichkeit  wurde  von  McGollum 
und  Hart1)  in  neuerdings  mitgeteilten  Versuchen  iiber  Ver- 
fiitterung  von  « dissected  milk»  erwogen.  Weiter  ist  es  notig, 
mit  der  Moglichkeit  des  Fehlens  einiger  spezifischer  Wachstums- 
« Hormone »  zu  rechnen,  welche  vielleicht  in  der  eiweiBfreien 
Milch  vorhanden  sind  und  sie  zur  Erregung  des  Wachstums 
unter  anderen  giinstigen  Bedingungen  geeignet  machen.  Hat 
nicht  Stepp2)  behauptet,  daB  eine  Erhaltung  unmoglich  ist 
ohne  die  Gegenwart  von  lipoidahnlichen  Substanzen  in  der 
Nahrung?  Die  angebliche  wachstumsfordernde  Eigenschaft  des 
Lecithins  ist  breit  verhandelt  worden.  Der  Wert  kleiner  Bei- 
mischungen  fur  die  Unterhaltung  des  Wachstums  ist  in  ahn- 
licher  Weise  von  Funk3)  angedeutet  worden  in  neuen  Unter- 
suchungen  iiber  die  im  geglatteten  Reis  enthaltenen  Substanzen, 
die  zur  Behandlung  der  peripheren  Neuritis  geeignet  sein  sollen 
und  an  Vogel  verfuttert  worden  sind. 

Die  von  uns  in  dieser  Richtung  gemachten  Schlusse  sind 
deshalb  berechtigt,  weil  die  Bestandteile  der  Nahrung  mit  Aus- 
nahme  der  eiweiBfreien  Milch  in  jedem  Falle  sorgfaltigst  isoliert 
und  gereinigt  waren.  Bei  Fiitterung  mit  eiweiBfreier  Milch 
war  irgend  ein  atherloslicher  Faktor  als  Wachstumsforderer 
leicht  dadurch  auszuschlieBen,  daB  die  eiweiBfreie  Milch  vorher 
langere  Zeit  mit  Ather  extrahiert  worden  war.4)  Wenn  das 
in  dieser  Weise  vorbehandelte  Produkt  mit  einem  geeigneten 
EiweiBkorper  angewandt  wurde,  trat  auch  keine  Wachstums- 
hemmung  ein,  wie  die  folgenden  Kurven  zeigen. 

E.  V.  Mc  Coll  urn  and  E.  B.  Hart,  Journal  of  biological  che- 
mistry, 1912,  Bel.  11,  S.  XV. 

*)  W.  Stepp,  Biochemische  Zeitschrift,  1909,  Bd.  22,  S.  452;  Zeit- 
schrift  fur  Biologie,  1911,  Bd.  57,  S.  135. 

3)  Funk,  Journal  of  Physiology,  1911,  Bd.  43,  S.  395. 

4)  Der  Atherextrakt  der  vollstanclig  trockenen  proteinfreien  Milch 
betragt  nur  0,13  °/o. 


354        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


160 


wo- 


rn 


60 


/ 

— /  ✓ 

J  / 

/, 

/ 

r  I 
/  j 

/  / 
/  / 

/  / 

/  / 

/ 

/ 

y 

r 

Ei  we/SSL 
//  -fre/e 
Case/r, 

inaf 

VO         60         60  100 

fege 

Kurve  54. 


120 


100 


1 

r 

7 

/ 

1 

r 

1 

i  r 
I  j 
I 

J—J— 

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t  J 

7 

>'ss  //  fe/f 
£c/es> 

y/7 

20         W         60  80 


Kurve  55. 


Kurve  54  (Ratte  373  </)  Gaseinfiitterung,  Kurve  55 
(Ratte  375  <?)  Edestinfiitterung,  zeigen  entsprechendes  Wachs- 
tum  bei  Verwendung  der  mit  Ather  extrahierten  « eiweiBfreien 
Milch ». 


Nahrung: 

Ratte  373  Ratte  375 

°/o  °/o 

Casein  (Kuh                                                 18,0  0,0 

Edestin  (Hanfsamen)                                       0,0  18,0 

Mit  Ather  extrahierte  «eiweiftfreie  Milch*          28,0  28,0 

Starke                                                         21,0  19,0 

Agar                                                          5,0  5,0 

Fett                                                          28,0  30,0 


Ein  in  dieser  Beziehung  gleiches  Resultat  erhielten  wir 
durch  weiter  unten  erwahnte  Versuche,  in  welchen  wir  eine 
vollig  fett-  und  lipoidfreie  Nahrung  verfutterten.  Weitere 
Forschungen  nach  den  hypothetischen  Wachstumshormonen  der 
eiweiBfreien  Milch  wurden  nach  anderer  Richtung  angestellt. 


Uber  Fiitterungsversuche  mit  isolierten  Nahrungssubstanzen.  355 


Nachdera  wir  fanden,  daB  wir  die  Phosphate  und  das  Calcium 
der  eiweiBfreien  Milch  niederschlagen  und  sie  durch  das  reine 
kunstliche  Calciumphosphat  ohne  irgend  einen  Nachteil  fur  das 
Wachstum  des  Tieres  ersetzen  konnen,  haben  wir  versucht, 
die  Zusammensetzung  der  eiweiBfreien  Milch  durch  eine  kunst- 
liche Synthese  genau  nachzuahmen. 

Eine  Mischung  von  krystallinischen  Salzen,  welche  die 
verschiedenen  in  der  eiweiBfreien  Milch  vorhandenen  Ionen 
enthalt,  wird  von  der  eingefiihrten  Nahrung  in  ungleichmaBiger 
Weise  aufgenommen.  Dadurch  erfolgt  im  Darminhalt  wahrend 
des  Verdauungsprozesses  eine  Mischung  von  Ionen,  welche  von 
der  Mischung,  wie  sie  ursprunglich  in  der  Milch  vorhanden 
ist,  vollstandig  differiert.  Wir  bemiihten  uns,  diese  Schwierig- 
keit  dadurch  zu  umgehen,  daB  wir  eine  kunstliche  eiweiBfreie 
Milch  darstellten.  Bei  der  Darstellung  einer  Quantitat,  die  fur 
1  kg  Nahrung  reichen  sollte,  losten  wir  in  ca.  450  ccm  Wasser 
12,75  g  HC1,  10,32  g  H3P04,  10,10  g  CcH807 .  H20  (Acidum 
citricum)  und  0,92  g  H2S04.  Zu  dieser  Losung  fugten  wir  hinzu 
13,48  g  CaC03,  2,42  g  MgC03,  und  nachdem  diese  Salze  sich 
gelost  hatten,  eine  Losung  von  14,13  g  K2G03,  14,04  g  Na2C03 
und  0,634  g  FeC6H507  +  Vh  g  H20  in  ca.  100  ccm  Wasser. 
Zu  der  milchigen,  leicht  alkalisch  reagierenden  Flussigkeit 
wurden  246  g  Milchzucker  hinzugefiigt  und  die  Mischung  bei 
etwa  70°  verdampft.  Wahrend  des  Erhitzens  loste  sich  der  Milch- 
zucker, so  daB  eine  gleichformige  Mischung  all  dieser  Sub- 
stanzen  beim  Trocknen  erfolgte.  Beim  Verdampfen  zur  Trocken- 
heit  wurde  die  Mischung  dem  neutralisierten  Milchserum  ahnlich, 
welches  wir  in  derselben  Weise  bei  der  Darstellung  der  eiweiB- 
freien Milch  verdampft  hatten.  Die  verwandten  Salze  wurden 
alle  sorgfaltigst  analysiert  und  in  jeder  Weise  in  acht  genommen, 
um  die  genaue  Zusammensetzung  der  anorganischen  Bestand- 
teile  der  eiweiBfreien  Milch  zu  erhalten.  Entsprechend  dieser 
Analyse  enthielt  die  « eiweiBfreie  Milch »  in  1kg  der  Nahrung: 

Ca  5,9  g 

Mg  0,7  » 

Na  6,1  » 

K  8,0  » 

Hoppe-Seyler's  Zeitschrift  f.  physiol.  Chemie.  LXXX. 


356 


Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


P04  10,0  g 

CI  12,4  » 

S04  0,9  » 

Acid,  citric.1)    10,1  » 

Fe1)  0,13  » 

Das  eben  beschriebene  Produkt  ist,  den  physikalischen 
Eigenschaften  nach,  der  natiirlichen  eiweififreien  Milch  uber- 
raschend  ahnlich.  Wie  erfolgreich  sie  die  letztere  in  der  Nahrung 
vertreten  undersetzen  kann,  zeigen  einige  folgende  Aufstellungen : 

Die  Kurven  zeigen  das  Wachstum  bei  einer  Diat,  welche 
kiinstliche  Mischung  von  anorganischen  Salzen  enthalt. 


T*9e  Tege 


Kurve  56.  Kurve  57. 

Kurve  56  (Ratte  490  <?)  und  Kurve  57  (Ratte  553  ?) 
zeigen  das  Wachstum  bei  einer  Diat,  in  welcher  nur  kiinstliche 
Mischungen  von  anorganischen  Salzen  angewandt  wurden. 


*)  Letztere  beide  nach  der  Analyse  der  Milchasche  geschatzt. 


Uber  Fiitterungsversuche  mit  isolierten  Nahrungssubstanzcn.  357 


Nahrung: 


Casein  (Kuh) 

«Kiinstliche  eiweififreie  Milch » 

Starke 

Fett 


v/0 

18,0 
29,5 
26,5 
26,0 


Dies  sind,  soviel  wir  wissen,  die  ersten  erfolg- 
reichen  Fiitterungsversuche,  in  welchen  ein  an- 
dauerndes  Wachstum  mit  sorgfaltig  gereinigten  Nah- 
rungsstoffen  und  kiinstlichen  Salzmischungen  erreicht 
vvurde. 

Wachstum  bei  einer  von  atherloslichen  Substanzen  freien  Diat. 
Wir  sind  in  unserem  Bemiihen,  die  Versuchsbedingungen, 
unter  welchen  Ernahrung  und  Wachstum  studiert  werden 
konnen,  zu  vereinfachen,  noch  einen  Schritt  weitergegangen. 
Das  Fett  wurde  vollstandig  aus  der  Ernahrung  eliminiert  und  ein 
selbstandiges  Wachstum  der  Ratten  durch  eine  aus  einem  einzigen 
isolierten  Eiweifikorper,  ferner  aus  Kohlenhydraten  und  einer 
kiinstlichen  Salzmischung  bestehenden  Diat  hervorgerufen.  Ein 
einzelner  Versuch  folgt.  Die  Einzelheiten  dieser  Untersuchungs- 
richtung  miissen  fur  eine  besondere  Gelegenheit  aufgehoben 
werden. 

DaB  die  Tiere  ohne  UberfiuB  von  Fett  in  der  Nahrung 
wachsen  konnen,  ist  an  sich  nicht  iiberraschend.  Rosenstern 
bemerkt:  «Der  Fetthunger  ist  praktisch  von  geringer  Bedeutung, 
ist  es  doch  moglich,  Sauglinge  monatelang,  ohne  daB  die  ge- 
ringsten  Storungen  auftreten,  fast  vollkommen  fettfrei  zu  er- 
nahren,  wofern  nur  der  Energiebedarf  gedeckt  ist  —  eine  in 
Anbetracht  des  hohen  Fettgehalts  der  Frauenmilch  von  vorn- 
herein  merkwurdige  Tatsache».1)  Aber,  daB  ein  Wachstum 
bei  vollstandigem  Fehlen  jeder  Spur  einer  atherloslichen  Sub- 
stanz  moglich  sei,  ist,  soweit  uns  bekannt,  noch  nicht  experi- 
mentell  gezeigt  worden  und  ist  tatsachlich  das  Gegenteil  von 
dem,  was  Stepps  Versuche  an  Mausen  gezeigt  haben.2) 

1)  J.  Rosenstern,  Ergebnisse  der  inneren  Medizin  und  Kinder- 
heilkunde,  1911,  Bd.  7,  S.  390. 

2)  W.  Stepp,  Biochemische  Zeitschrift,  1909,  Bd.  22,  S.  452;  Zeit- 
schrift  fur  Biologie,  1911,  Bd.  57,  S.  135. 


Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Kurve  58  (Ratte  529  <?)  zeigt  das  Wachstum  bei  Ver- 
abreichung  einer  von  atherloslichen  Substanzen  freien 
Nahrung,  in  welcher  die  anorganischen  Salze  in  kunstlich 
hergestellten  Mischungen  angewandt  wurden. 


700 


180 


160 


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Kurve  58. 


Die  Unterdriickung  des  Wachstums  und  die  Fahigkeit,  das  Wachstum 
wieder  aufzunehmen. *) 

Wie  lange  kann  die  Wachstumskapazitat  zuriickgehalten 
werden  und  kann  sie,  wenn  sie  einmal  unterdruckt  worden 
ist,  zu  ihrer  vollen  GroBe  wieder  hergestellt  werden?  Minot2) 
hat  angedeutet,  daB  die  Schnelligkeit  des  Wachstums  von  der 

l)  Cf.  T.  B.  Osborne  und  L.  B.  Mendel,  Carnegie  Institution  of 
Washington,  Publication  156,  Part  II,  S.  71. 

8)  Minot,  Alter,  Wachstum  und  Tod.    New  York  1908,  Chap.  III. 


Uber  Futterungsversuche  mit  isolierten  Nahrungssubstanzen.  359 


Jugend  des  Individuums  abhiingt  —  der  zeitlichen  Entfernung 
von  seiner  Geburt.  Rubner  driickt  folgende  Ansichten  aus: 
«Wir  wissen  eigentlich  gar  nicht,  ob  die  Natur  ein  absolut 
gleichmaBiges  tagliches  Wachstum  verlangt,  oder  ob  Reraissionen 
zulassig  oder  gar  zweckmaBig  sind.  Nur  das  steht  sicher,  daB 
die  Behinderung  des  Wachstumstriebes,  wie  dies  wirklich  vor- 
koramt,  nicht  wahrend  der  ganzen  Wachstumsperiode  andauern 
darf,  da  sonst  die  GroBe  des  Individuums  dauernd  Schaden 
leidet.  Verlorene  KorpergroBe  in  der  Jugendzeit  kann  nach 
Vollendung  der  Wachstumsperiode  nimmermehr  abgeglichen 
werden*.1) 

In  unseren  Unter- 
suchungen  haben  sich 
die  Daten  uber  diese 
Fragen  gemehrt.  DaB 
eine  Unterdriickung  des 
Wachstums  fur  kurze 
Perioden  von  einer 
volligen  Wiederherstel- 
lung  gefolgt  sein  kann, 
ist  wohl  bekannt.  Das 
wird  veranschaulicht  in 
den  folgenden  Kurven, 
in  welchen  das  Wachs- 
tum nach  einem  Still- 
stand  infolge  ungeeig- 

neter  Salzmischungen  (Kurve  59)  oder  ungeeigneter  EiweiB- 
korper  im  Futter  wieder  aufgenommen  wurde  (Kurve  60). 

Kurve  59  (Ratte  189  ?)  zeigt  die  Wiederaufnahme  des 
Wachstums  nach  einem  2  Monate  dauernden,  durch  nicht  ent- 
sprechende  Kohlenhydrate  und  Salzmischungen  des  Futters 
verursachten  Gewichtsabfall. 


160 


120 


WO 


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c 

/ 



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V/7 

-fnefeAf/. 

0*  -X- 

-> 

r 

2 

20 


uo 


60 
Tage 

Kurve  59. 


80 


too 


*)  Rubner,  Archiv  fiir  Hygiene,  1908,  Bd.  66,  S.  82. 


360 


Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Nahrung: 

Periode  1  Periode 

o/o  O/o 

Edestin  (Hanfsamen)           18,0  0,0 

Milchpulver                         0,0  60,0 

Starke                             29,5  15,7 

Zucker                             15,0  0,0 

Salzmischung  I                    2,5  1,0 

Agar                                 5,0  0,0 

Fett                                30,0  23,3 


120 


60 


4 

f 

/ 

/ 

/ 

t 

// 

/  y 
/  / 

Weizen- 
Viadin  - 

// 

_  _  a 

-  -> 

7 

IS 

20 


to 


60 


120 


160 


180 


Kurve  60. 


Kurve  60  (Ratte  381  ?)  zeigt  die  Wiederaufnahme  des 
Wachstums  nach  einer  lange  fortgesetzten,  durch  nicht  ent- 
sprechendes  EiweiB  verursachten  Unterdriickung  desselben. 
Beweis,  daB  das  wieder  aufgenommene  Wachstum  durch  Ver- 
futterung  einer  Mischung  von  isolierten  Nahrungssubstanzen 
verursaeht  wurde. 

Nahrung: 


Periode  2 

Periode  3 

°/o 

7° 

Gliadin  (Weizen) 

18,0 

0,0 

Casein  (Kuh) 

0,0 

18,0 

«EiweiMreie  Milch* 

28,2 

28,2—28,0 

Starke 

20,8 

23,8—27,0 

Agar 

5,0 

5,0—0,0 

Fett 

28,0 

25,0-27,0 

Uber  FiUterungsversuche  mit  isolierten  Nahrungssubstanzen.  301 


Die  Wiederaufnahme  des  unterdruckten  Wachstums  infolge 
quantitativer  Unzulanglichkeit  der  Ernahrung  wird  in  ahnlicher 
Weise  ira  folgenden  gezeigt. 


f  s 

y 

*ase/n 

f8% 

/ 

/ 

/ 

£  Casein 

/ 

/ 

/  /"" 
/  / 

1  J 

/ 

2 

20  W         60  80         iOO       120        I'M        160  180 

Kurve  61. 


Kurve  61  (Ratte  340  ?)  zeigt  die  Wiederaufnahme  des 
Wachstums  nach  einer  Unterdnickung  desselben  infolge  quan- 
titativer Unzulanglichkeit  der  EiweiBkorper  in  der  Diat.  Der 
wesentliche  Unterschied  in  den  Ernahrungsformen  der  zwei 
Perioden  besteht  im  Gehalt  an  Casein.  Ahnliche  Resultate 
zeigt  die  Kurve  45,  in  welcher  das  Wachstum  nach  einer  uber 
100  Tage  dauernden  wachstumslosen  Periode  wieder  aufge- 
nommen  wurde. 

Nahrung: 


Periode  1 

Periode  2 

°/o 

°/o 

Casein  (Kuh) 

12,0 

18,0 

«EiweiMreie  Milch» 

28,0 

28,0 

Starke 

22,0 

22,0—27,0 

Lactose 

6,0 

0,0 

Agar 

5,0 

5,0-0,0 

Felt 

27,0 

27,0 

362        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


Die  Perioden,  nach  welchen  eine  Wiederaufnahme  des  unter- 
brochenen  Wachstums  erfolgt,  sind  hier  verhaltnismaBig  kurz. 
GroBeres  Interesse  verdient  es,  wenn  der  Wachstumsstillstand 
bis  zu  einem  Alter  ausgedehnt  wird,  in  dem  gewohnlich  kurze 
Zeit  spater  das  Wachstum  der  Tiere  vollendet  ist.  DaB  der 
Wachstumsimpuls  in  diesem  spateren  Alter  nicht  erloschen  isl, 
zeigen  die  Kurven  62  und  63,  in  welchen  in  einem  Alter  von 
237  und  304  Tagen  ein  erneutes  normales  Wachstum  erfolgte. 

Kurve  62  (Ratte  196  ?)  zeigt  die  Wachstumskapazitat 
nach  einer  177  Tage  dauernden  Wachstumsunterbrechung.  Die 
einzigen  Unterschiede  in  der  Diat  bestanden  in  dem  Ersatz 
der  anorganischen  Salzmischung  und  des  Zuckers  der  Periode 
1  bis  4  durch  die  eiweiBfreie  Milch  der  Periode  4. 


Nahrung: 

Periode  1,2,3 

Periode  4 

°/o 

•jo 

Edestin  (Hanfsamen) 

18,0 

18,0 

«Eiweiftfreie  Milch* 

0,0 

28,2 

Starke 

29,5 

25,8—20,8 

Zucker 

15,0 

0,0 

Agar 

5,0 

0,0—5,0 

Salzmischung  I 

2,5 

0,0 

Fett 

30,0 

28,0 

Uber  andere  Versuche,  welche  die  Wachstumskapazitat 
nach  bedeutend  kurzeren  Perioden  von  Wachstumsunterbrechung 
demonstrieren,  siehe  «Futterungsexperimente  mit  isolierten  Nah- 
rungssubstanzen»  Carnegie  Institution  of  Washington,  Publi- 
cation 156,  Part  II,  p.  92,  93,  98,  103,  109,  112,  113,  122, 
123,  124,  133,  134;  Charts  XXVIII,  XXIX,  XXXVII,  XL VI, 
XLVII,  LXV,  LXXI,  LXXII,  LXXVIII,  XCVI,  XCVII,  XCIX,  C, 
CXX,  CXXI,  CXXII,  CXXIII. 

Kurve  63  (Ratte  240  ? )  zeigt  die  Wachstumskapazitat  nach 
einem  fur  265  Tage  durch  die  Verfiitterung  von  Gliadin,  als 
alleinigem  EiweiBkorper  der  Nahrung,  unterbrochenen  Wachs- 
tum. 


tiber  Futterungsversuche  mil  isolierten  Nahrungssubslanzen. 


363 


Thomas  B.  Osborne  und  Lafayette  B.  Mendel. 


Nahrung: 


Periode  1 

Periode  2 

Gliadin  (Weizen) 

°/0 

18.0 

% 
0,0 

Milchpulver 

0,0 

60,0 

«EiweiGfreie  Milch* 

28,2 

0,0 

Starke 

20,8 

16,0 

Agar 

5,0 

0,0 

Fett 

28,0 

24,0 

Solche  Erfolge  miis- 
sen  scharf  von  derWie- 
derernahrung  solcher 
Tiere  unterschieden 
werden,  die  zunachst 
Gewichtsverluste  erlit- 
ten  haben.  Die  Wieder- 
herstellung  desGewichts 
i.  e.  Wiederernahrung. 
mag  unter  Bedingungen 
erfolgen,  unter  welchen 
ein  Wachstum  unmog- 
lich  ist. 

Kurve  64  (Ratte 
147  ?)zeigtdie  Wieder- 
herstellung  desGewichts 
in  Periode  2  nach  einem 
Gewichtsabfall  in  Perio- 
de 1.  Dieser  Typus  der 
Wiederherstellung  muB  unterschieden  werden  von  der  Gewichts- 
zunahme,  die  fur  ein  wirkliches  Wachstum  charakteristisch  ist 
und  durch  Gliadin  nicht  veranlaBt  werden  kann. 

Nahrung: 


Li 



«J  .UJ 

■  i 

_  We/ze 

7  -G/iad 
s-fre/e 

n  

'  s  \ 

-\ 

Z 

-v  

\ 

\ 

V.  

V 

> 

20 


60 

rage 

Kurve  64. 


too 


J2o 


Periode  1 

Periode  2 

0/0 

7° 

Gliadin  (Weizen) 

18,0 

18,0 

«EiweiBfreie  Milch > 

0,0 

28,2 

Starke 

29,5 

20,8—25,8 

Zucker 

17,0 

0,0 

Agar 

5,0 

5,0-0.0 

Salzmischung  I 

2.5 

0,0 

Fett 

28.0 

28.0 

Uber  Futterungsversuche  mit  isolierten  Nahrungssubstanzen.  365 

Einige  Bemerkungen  und  SchluBfolgerungen. 

Der  Erfolg,  welcher  die  im  Vorhergehenden  wiederge- 
gebenen  Versuche  begleitet  hat  und  der  darin  besteht,  daB  es 
gelang,  bei  Tieren  ein  charakteristisches  Wachstum  durch  iso- 
lierte  Nahrungssubstanzen  zu  erzielen,  ofTnet  den  Weg  fur  wert- 
vollere  Forschungen  liber  die  zahlreichen  individuellen  Faktoren 
im  Wechsel  der  Entwicklung.  Es  hat  sich  gezeigt,  daB  es  mog- 
lich  ist,  ein  junges  Tier  durch  den  groBten  Teil  seiner  selbst- 
tatigen  Wachstumsperiode  zu  Ziehen,  wahrend  welcher  Zeit  sein 
Korpergewicht  sich  unter  Darreichung  einer  Mischung  von  sorg- 
faltig  gereinigten  EiweiBkorpern,  Starke.  Zucker,  Fett  und  an- 
organischen  Salzen  mehrfach  vervielfacht.  Wenn,  wie  es  mog- 
lich  erscheint,  diese  Mischung  insofern  weiter  vereinfacht  werden 
kann,  daB  die  Fette  und  alle  anderen  atherloslichen  Substanzen 
(in  bezug  auf  Reinheit  die  unsichersten  Komponenten)  aus- 
geschaltet  werden.  sind  die  chemischen  Ernahrungsprobleme 
einer  erfolgreichen  experimentellen  Losung  um  so  naher  geriickt. 

Rosenstern  hat  kurzlich  geschrieben:  «Wenn  man  bis- 
lang  noch  nicht  imstande  ist,  einen  Organismus  mit  einem  kiinst- 
lichen  Nahrgemisch  am  Leben  zu  erhalten,  so  spielt  dabei  u.  a. 
wohl  ein  Mangel  an  Reizstoffen  eine  Rolle,  auf  deren  Bedeutung 
die  Pawlowschen  Untersuchungen  ein  Licht  geworfen  haben.* x) 
Abgesehen  von  irgend  einer  Wirkung,  wrelche  die  Nahrstoffe 
an  sich  und  die  anorganischen  Salze  haben,  kann  ein  beson- 
derer  «Reiz»-Faktor  jedoch  kaum  als  wesentlich  fiir  den  nutri- 
tiven  Erfolg  in  Betracht  kommen.  Vage  Ansichten  iiber  solche 
hypothetische  Komponenten  sollten  uns  aber  nicht  langer  ver- 
wirren. 

Die  experimentellen  Daten  dieser  Arbeit  haben  gezeigt, 
was  zu  erwarten  war,  daB  namlich  ein  gewdsses  Minimum  an 
EiweiBzufuhr  fur  das  Wachstum  notig  ist.  Betrage  von  EiweiBzu- 
fuhr,  die  unterhalb  des  Wachstumsbedarfs  liegen,  sind  —  gleichen 
Energieersatz  vorausgesetzt  —  natiirlich  keineswegs  mit  einer 
Erhaltung  des  Lebens,  sogar  ohne  Gewichtsverlust,  unverein- 

*)  J.  Rosenstern,  Ergebnisse  der  inneren  Medizin  und  Kinder- 
heilkunde,  1911,  Bd.  7,  S.  393. 


366         Thomas  B.  Osborne  und  Lafayette  B.  Mendel. 

bar.  Es  besteht  Erhaltung  statt  Wachstum.  Diese  Unterscheidung 
wurde  von  anderer  Seite  als  «Betriebsstoffwechsel»  und  «Baustoff- 
wechsel»  ausgedriickt.  Es  ist  wichtig,  daB  ein  verhaltnismaBig 
kleiner  Betrag  an  EiweiB  geniigt,  um  ein  ausreichendes  Wachs- 
tum zu  unterhalten ;  ein  starkeres  Wachstum  kann  durch  uber- 
maBige  EiweiBzufuhr  nicht  hervorgerufen  werden.  Ahnliche 
Resultate  ergaben  sich  in  unseren  Versuchen  auch  im  Hinblick 
auf  das  Optimum  und  Minimum  der  anorganischen  Bestandteile 
der  Nahrung. 

Weiterhin  interessierte  der  EinfluB,  welchen  die  qualita- 
tiven  Eigenschaften  der  EiweiBkorper  und  Salze  auf  das  Wachs- 
tum ausiiben.  Die  Zahl  der  EiweiBkorper,  welche  sich  als  von 
giinstigem  EinfluB  auf  das  Wachstum  erwiesen  haben,  ist  nicht 
gering.  Sie  umschlieBt  EiweiBkorper  verschiedenster  Herkunft 
und  zweifelsohne  verschiedenster  Zusammensetzung.1) 

Tafel  I  zeigt  die  Gewichtszunahme  (oberhalb  des  Null- 
punkts)  und  die  Gewichtsabnahme  (unterhalb  des  Nullpunkts) 
wahrend  der  ersten  30  Tage,  in  welcher  Zeit  mit  verschiedenen 
EiweiBkorpern  sowohl  tierischen  wie  pflanzlichen  Ursprungs 
gefuttert  wurde.  Die  unter  Phaseolin-  und  Leimernahrung 
verzeichneten  Gewichtsverluste  sind  nur  geschatzt,  da  die 
Versuche  mit  diesen  Proteinen  nicht  durch  30  Tage  hin- 
durch  gefuhrt  werden  konnten  wegen  der  groBen  Gewichts- 
verluste der  jungen  Tiere.  Die  groBten  Gewichtsverluste,  welche 
unter  Phaseolinfutterung  gezeigt  werden,  erfolgten  bei  zwei  Ratten 
von  ca.  50  g  Anfangsgewicht,  die  kleineren  bei  Ratten  von 
ca.  35  g  Anfangsgewicht. 

Bei  Betrachtung  der  Unterschiede  zwischen  diesen  ein- 
zelnen  Versuchsreihen  muB  in  Betracht  gezogen  werden,  daB 
diese  Versuche  in  ihrer  Anordnung  neu  sind  und  an  groBeren 
Zahlen  von  Tieren  wiederholt  werden  miissen. 

Worin  die  Unzulanglichkeit  mancher  dieser  EiweiBkorper 
liegt,  sind  wir  nicht  imstande  zu  sagen.    Es  muB  hervorge- 

l)  Fiir  Angaben  iiber  die  verschiedenen  Typen  von  N-Kombinationen 
in  den  meisten  dieser  Eiweiftkorper  siehe  T.  B.  Osborne,  C.  S.  Leaven- 
worth and  C.  A.  Brautlecht,  American  Journal  of  Physiology,  1909, 
Bd.  23,  S.  180. 


Uber  Futtcrungsversuche  mit  isoliertcn  Nahrungssubstanzen.  3 


hoben  werden,  daB 
durch  irgend  einen 
EiweiBkorper ,  dem 
die  zyklische  Gruppe, 
wie  sie  im  Tyrosin 
und  Tryptophan  ge- 
fundenwurde,1)  fehlt, 
ein  Wachstum  nicht 
vollendet  werden 
kann.  W.  A.  Os- 
borne hat  in  der 
Tat  die  Hypothese 
aufgestellt,  daB  die 

«Cyclopoiese»  in 
diesem  Sinne  eine 
Eigentiimlichkeit  der 
pflanzlichen  Zelle  ist, 
und  daB  der  tierische 
Organismus « acyclo- 
poietisch»  und  fiir 
gewisse  Typen  seiner 
Nahrung  vom  pflanz- 
lichen Leben  ab- 
hangig  ist.  Diese  un- 
wirksamen  EiweiB- 
korper sind  aber 
nicht  urspriinglich 
toxisch,  das  erhellt 
aus  der  Tatsache, 
daB  manche  unter 
ihnen  sich  als  durch- 
aus  ausreichend  fiir 
die  Erhaltung  des  Ge- 
wichtsstillstandes  bei 


*)  Gf.auchE.  Abder- 
h  al  d  e  n ,  Diese  Zeitschr., 
1912,  Bd.  76,  S.  22. 


Hon  *e/n 
Z_  ]  £n  'sen 


Phas^o/rn 

I  lew 
\Leim 

\  Konghjrm 


\Rogg 


'n-g/t'aitn 


■g  f/aaf/n 


\u6. 


Safe. 


///? 


Ma/s  y/u/e///? 

£xce/s//7 

/(urd/ssa/nen- 
g /o fa ///'/?■ 


Ova  t6u/r?/n 


odse/n 


\Uvo- 


368        Thomas  B.  Osborne  und  Lafayette  B.  Mendel, 


wachsenden  und  erwachsenen  Tieren  erwiesen  haben,  wahrend, 
wie  bekannt,  andere,  so  Zein  und  Leim,  zu  diesem  Behufe  nicht 
genugen.  Ferner  geniigen  Beimengungen  von  kleinen  Mengen 
eines  wirksamen  EiweiBkorpers  zur  Anregung  des  Wachstums 
bezw.  Erhaltung  des  gleichen  Gewichts.1) 

Trotz  des  normalen  Charakters,  der  aus  manchen  unserer 
erfolgreicheu  Versuche  entnommenen  Wachstumskurven  sind 
wir  noch  nicht  imstande,  unter  den  kiinstlich  geschaffenen  Be- 
dingungen  eine  vollstandig  normale  Entwicklung  hervorzurufen. 
Wenn  wir  an  die  Ernahrung  des  Kindes  denken,  so  fallt  uns 
auf,  daB  manches  Kind  eine  ausreichende  Gewichtszunahme 
zeigt,  dabei  aber  offenbar  anamiseh  ist  und  eine  schlecht  ent- 
wickelte  Muskulatur  hat.  Wenn  ein  solches  Kind  Anstrengungen, 
die  tiber  das  physiologische  MaB  hinausgehen,  ausgesetzt  wird, 
dann  kann  es  in  einer  Weise,  die  bei  dem  wirklich  normalen 
Kind  nicht  moglich  ist,  versagen. 

Ein  Beispiel  dafur  ist  die  klinische  Beschreibung  eines 
kiinstlich  ernahrten  Kindes:  «Der  Zustand,  in  dem  sich  die 
Kinder  dabei  finden,  ist  aber  zweifellos  nicht  als  ein  normaler 
zu  bezeichnen,  das  zeigt  sich,  wenn  aus  therapeutischen  Griinden 
ein  Hunger  eingeleitet  wird  oder  wenn  eine  Ernahrungsstorung 
das  Gedeihen  unterbricht.  Es  treten  dann  in  kurzer  Zeit  bis- 
weilen  ganz  rapide  Gewichtsverluste  ein,  begleitet  von  schweren 
Allgemeinerscheinungen. » 2) 

Chemische  und  histologische  Untersuchungen  der  bei 
den  Experimentaltieren  vorhandenen  Gewebe,  Ziichtungs- 
versuche,  Immunitatsversuche  und  andere  Untersuchungen 
sind  notig,  um  Licht  in  die  Materie  zu  bringen.  Der  Ein- 
fluB  von  spezifischer  Ernahrung  auf  die  Hauptdrusen,  welche 
bekanntlich  eine  wichtige  Bolle  innehaben,  ist  zugleich  zu  er- 
forschen.  Es  ist  z.  B.  moglich,  daB  der  durch  manche  unserer 
dargereichten  Nahrungsstoffe   verursachte  Gewichtsstillstand 

*)  Zur  Veranschaulichung  dieser  Tatsache  siehe  Karte  CXX— CXXIII 
in  unsere  Publication  156,  Carnegie  Institution  of  Washington,  1911, 
Part  II,  S.  133  u.  134. 

8)  Rosenstern,  Ergebnisse  der  inneren  Medizin  und  Kinderheil- 
kunde,  1911,  Bd.  7,  S.  393. 


Uber  Fiilterungsversuche  mit  isoliertcn  Nahrungssubstanzen.  369 

—  mag  er  durch  qualitativ  oder  quantitativ  schlechte  Ernahrung 
veranlaBt  sein  —  die  indirekte  Veranlassung  zu  einer  Unter- 
entwicklung  von  Thymus,  Thyreoidea,  der  Sexual-  oder  anderer 
Driisen  ist.  Es  ware  eine  interessante  Betrachtung,  die  Be- 
ziehungen  unserer  «Zwerge»  zum  menschlichen  Infantilismus1) 
zu  untersuchen. 

In  dieser  Beziehung  muB  hervorgehoben  werden,  daB  die 
Versuche  iiber  Gewichtsstillstand,  wie  er  durch  Gliadin  als 
EiweiB  veranlaBt  wurde  —  wobei  eine  Unterdruckung  des 
Wachstums  iiber  eine  verhaltnismaBig  lange  Periode  der  ab- 
geschatzten  Lebensdauer  des  Tieres  ausgedehnt  wird  — ,  eine 
giinstige  Gelegenheit  darbieten,  um  die  WachstumsgroBe  in  einer 
vollstandig  anderen  Weise  zu  untersuchen,  als  dies  die  Re- 
generation und  Wiederherstellung  ermoglicht. 

Mc  Gollum2)  hat  kiirzlich  klar  gezeigt,  daB  die  Wiederher- 
stellungsprozesse  sich  von  den  Wachstumsprozessen  in  ihrem 
Charakter  unterscheiden.  Nach  ihm  verursachen  die  Prozesse 
der  Zellerneuerung  keine  Zerstorung  und  Neusynthese  eines 
ganzen  Proteinmolekiils.  Hierin  liegt  vielleicht  das  Geheimnis, 
daB  die  sogenannten  «unvollstandigen  EiweiBk6rper»  imstande 
sind,  einen  Gewichtsstillstand  zu  erhalten,  wahrend  sie  zur 
Bildung  neuer  Gewebe,  d.  h.  zum  Wachstum  nicht  ausreichen. 
Es  ist  zu  bedenken,  daB  der  Wachstumsimpuls  nicht  leicht  er- 
lischt,  wie  angenommen  wird.  DaB  diese  Kraft  lange  Zeit  zu- 
ruckgehalten  werden  kann,  ist  in  anderer  Hinsicht  durch  die 
Erscheinung  der  verschiedenen  Formen  von  abnormem  Wachs- 
tum gezeigt  worden,  wie  Tumoren,  Garcinom  und  Sarcom,  welche 
allgemein  als  auf  Wachstum  bedachte  Zellen,  die  der  hindernden 
Kontrolle  entbehren,  aufgefaBt  werden.  Da  die  mit  Ratten  an- 
gestellten  Versuche  iiber  Gewichtsstillstand  Bedingungen  zeigen, 
unter  welchen  die  Zellen  in  mehr  oder  weniger  jungem  Zu- 
stande  gehalten  werden,  konnen  wir  verstehen,  warum  der 

1)  Gf.  G.  A.  Herter,  Infantilism,  New  York  1908;  G.  Peritz,  Der 
Infantilismus,  Ergebnisse  der  inneren  Medizin  und  Kinderheilkunde,  1911, 
Bd.  7,  S.  405. 

2)  E.  V.  McGollum,  American  Journal  of  Physiology,  1911,  Bd.  29, 

S.  215. 


370    Th.  B.  Osborne  u.  L.  B.  Mendel,  Uber  Fiitterungsversuche  usw. 

Wachstumsimpuls  nicht  erlischt.  Deshalb  miissen  in  dem  Ge- 
webe  bei  sorgfaltiger  Untersuchung  mehr  jugendliche  als  senile 
Eigenschaften  erwartet  werden. 

Zum  Schlusse  darf  die  Richtung  der  hier  angefiihrten  Ver- 
suche,  die  von  der  Frage  der  Synthese  im  tierischen  Korper 
handelt,  nicht  iibersehen  werden.  Die  Physiologen  waren  nicht 
geneigt,  die  Moglichkeit  einer  Synthese  von  Aminosauren  dem 
Korper  de  novo  zuzuschreiben.  Die  GewiBheit,  die  mit  Riick- 
sicht  auf  das  Glykokoll  erbracht  wurde,  schien  dann  auBerhalb 
jeden  Zweifels. J) 

Andere  weitere  Daten  weisen  in  die  Richtung  der  Syn- 
thesefahigkeit  der  tierischen  Zelle.2)  Die  synthetische  Bildung  von 
Glykokoll  bei  unseren  Ratten,  welche  mit  dem  glykokollfreien 
Casein  gefuttert  wurden,  muB  als  feststehend  angenommen 
werden.  Entweder  erfolgte  die  Synthese  in  ihren  Korperzellen 
oder  unter  der  Wirkung  der  Darmbakterien.  Und  wenn  wir 
uns  erinnern,  daB  alle  unsere  Versuchsfiitterungen  purinfrei  und 
einige  vollig  frei  von  organisch  gebundenem  Phosphor  sind,  wird 
die  Tatsache  der  Synthese  uns  auf  einmal  klar.  Phosphor- 
proteine  und  Nucleoproteine  miissen  in  solchen  Organismen 
ohne  Zweifel  durch  komplizierte  synthetische  Umwandlungen  ent- 
stehen.3)  Bis  zu  welcher  Ausdehnung  und  in  welchen  Richtungen 
diese  letzteren  moglich  sind,  muB  die  Zukunft  erschlieBen. 

1)  Cf.  Ringer,  Journal  of  Biological  Chemistry,  1911,  Bd.  10,  S.  327, 
und  Epstein  and  Bookman,  ibid.,  S.  353.  Tiber  die  friihere  Literatur 
wird  in  diesen  neuen  Arbeiten  berichtet. 

2)  Cf.  Knoop,  Diese  Zeitschrift,  1910,  Bd.  67,  S.  489;  Zentralblatt 
fur  Physiologie,  1910,  Bd.  24,  S.  815;  Embden  und  Schmitz,  Biochem. 
Zeitschrift,  1910,  Bd.  29,  S.  423;  1912,  Bd.  38,  S.  392;  Kondo,  ibid., 
S.  407;  Fellner,  ibid,  S.  414. 

3)  Cf.  E.  V.  McCollum,  American  Journal  of  Physiology,  1909, 
Bd.  25,  S.  120;  Abderhalden,  Diese  Zeitschrift,  1912,  Bd.  76,  S.  22; 
Fingerling,  Biochemische  Zeitschrift,  1912,  Bd.  38,  S.  448;  McCollum 
and  Halpin,  Journal  of  Biological  Chemistry,  1912,  Bd.  11,  S.  13. 


Verlag  von  J.  V.  Bergmanp  in  Wiesbaden. 


Lehrbuch  der  Physiologischen  Chemie 

von 

Olof  Hammarsten, 

ehcm.  o.  o.  Professor  der  med.  und  physiol.  Chemie  an  der  Universit'at  Upsala. 

Siebente  vbllig  umgearbeitete  Auflage. 

Preis  Mk.  23.—,  geb.  Mk.  25 AO. 

Das  Hammarstensche  Lehrbuch  wird  von  jedem  als  ein  sehr 
lieber,  unentbehrlicher  Freund  und  Ralgeber  begriifit,  so  oft  es  in  neuer 
Gestalt  erscheint.  Es  ist  ja  in  der  Tat  das  Lehrbuch  der  physiol.  Chemie, 
und  wird  es  wohl  noch  lange  bleiben.  Wenigstens  in  seiner  Art  als 
absolut  zuverlassiges  Hand-  und  Nachschlagewerk  und  zur  kritischen  Ein- 
fuhrung  in  bestimmte  Fragen.  An  der  Anordnung  ist  nichts  geandert, 
nur  ein  sehr  willkommenes  Namenregister  angefugt.  Dafi  die  Literatur 
soweit  als  irgend  moglich  bis  auf  die  letzte  Zeit  beriicksichtigt  ist,  braucht 
nicht  erwahnt  zu  werden.  Biochemisches  Zentralblatt. 

Analyse  des  Harns. 

Zum  Gebrauch  fur  Mediziner,  Checker  und  Pharmazeuten 
zugleich 

Elfte  Auflage  von  Neubauer-Hupperts  Lehrbuch. 

Bearbeitet  von 

A.  Ellinger-Konigsberg,  F.  Falk-Wien,  L.  Henderson-Boston,  j 
F.  N.  Schultz-Jena,  K.  Spiro-Strafiburg  und  W.  Wiechowski-Wien. 

J  /.  Hdlfte.  —  Preis  Mk.  15.—. 

....  Neu  ist  an  der  Bearbeitung  besonders  auch,  daft  jetzt  die 
qualitativen  und  quantitativen  Bestimmungen  sich  der  Besprechung  der 
einzelnen  Stoffe  unmittelbaj  anschliefien,  was  fur  den,  der  nach  dem 
Buche  arbeiten  will,  entschieden  eine  grofie  Eiieichterung  darstellt.  Der 
Inhalt  des  Buches  ist  zu  reichhaltig,  um  auf  Einzelheiten  einzugehen, 
lafit  aber  nirgends  Vollstandigkeit  und  Uebersichtlichkeit  vermissen.  Die 
Autoren  durfen  ihr  Werk  der  Oeffentlichkeit  ubergeben  in  dem  BewuBt- 
sein,  einem  dringenden  Bedurfnis  entsprochen  und  Mustergiiltiges  geleistet 
zu  haben.  Zentralblatt  f.  Innere  Medizin. 

Dynamisehe  Bioehemie 

Chemie  der  Lebensvorgange. 

Von 

Professor  Dr.  Sigmund  Frankel,  Wien. 

Preis  Mk.  18.60,  gebunden  Mk.  20.20. 

Gewissermaften  als  zweiter  Band  zu  des  gleichen  Autors  «Deskrip- 
tiver  Bioehemie »  folgt  diese  dynamisehe  Bioehemie,  in  welcher  das  Haupt- 
gewicht  auf  das  chemische  Geschehen  im  Organismus  gelegt  wird.  In 
sehr  geschickter  Weise  wird  das  weitschichtige  Gebiet,  welches  ja  den 
grofieren  und,  abgesehen  vom  Kreislauf,  praktisch  wichtigsten  Teil 
der  Lebensvorgange  umfaftt,  dargestellt.  Fiir  die  Lesbarkeit  des 
Werkes  ist  es  wohl  ein  Vorzug,  dafi  der  Autor  in  der  Auswahl  des  zu 
besprechenden  Stoffes  eine  gewisse  Beschrankung  sich  auferlegt  hat. 

Deutsche  mediz.  Wochenschrift. 


Verlag  von  KARL  J.  TRUBNER  in  Strafibnrg. 


Im  September  gelangt  zur  Ausgabe: 

Chemie  der  Fette 

vom  physiologisch-chemischen 
Standpunkte. 

Von 

Prof.  Dr.  Adolf  Jolles, 

in  Wien. 


Zweite  vermehrte  und  verbesserte  Auflage. 


8°.  VII,  148  Seiten.  Geheftet  M  4.—,  in  Leinwand  geb.  Ji  4.50. 

Urteile  der  Presse  iiber  die  erste  Auflage: 

«Das  Werkchen  ist  eine  sehr  fleiftige  Sammlung  der  neueren  For- 
schungen  auf  dem  Gebiete  der  Fettchemie,  insoweit  sie  physiologisch 
bedeutsam  sind,  im  Zusammenhang  mit  den  in  Betracht  kommenden 
allgemeinen  Erfahrungen  der  organischen  und  physikalischen  Chemie.  Die 
Anzahl  der  die  wissenschaftlichen  Arbeiten  veroffentlichenden  Zeitungen 
und  Zeitschriften  ist  zu  einer  erstaunlicben  Hohe  angewachsen  und  macht 
es  dem  Einzelnen  schon  schwer,  selbst  auf  kleinerem  Gebiete,  alle  Ab- 
handlungen  und  Leistungen  zu  verfolgen.  Wer  nun  weifi,  welches  Mafi 
von  Miihe  und  Zeitaufwand  c-s  kostet,  bei  diesen  Mengen  von  Publikationen 
eine  so  vielfach  zerstreute  einschiagige  Literatur  zu  bearbeiten,  wird 
dieser  gewissenhaften  Zusammenfassung  Dank  wissen.> 

Osterreichische  Chemiker-Zeitung  1907,  Nr.  16. 

«.  .  .  Die  Arbeit  stellt  sich  als  ein  mit.  grofiem  Fleifi  zusammen- 
getragenes  Sammelreferat  dar,  welches  mit  seinen  zahlreichen  Literatur- 
angaben  jedem  willkommen  sein  mufi,  der  sich  theoretisch  oder  praktisch 
mit  der  physiologischen  Fettchemie  befafit  oder  sich  iiber  den  heutigen 
Stand  dieser  Wissenschaft  schnell  zu  orientieren  wunscht.» 

Apotheker-Zeitung  1907,  Nr.  95. 

<.  .  .  Das  Btichlein  wird  Chemikern  und  Physiologen  iiber  alle  auf 
die  Fette  bezuglichen  Daten,  Fragestellungen  und  Ergebnisse  eine  rasche 
Orientierung  ermoglichen.* 

Deutsche  Literaturzettung  1908,  Nr.  14. 


U.  Dn  Mont-Schauberg,  Strassburg.  —  365. 


Reprinted  fiom  The  Journal  of  Biological  Chemistry,  Vol.  XIII,  No.  3,  1912. 


THE  BEHAVIOR  OF  SOME  HYDANTOIN  DERIVATIVES 
IN  METABOLISM.  I. 
HYDANTOIN  AND  ETHYL  HYDANTOATE. 

By  HOWARD  B.  LEWIS. 

{From  the  Sheffield  Laboratory  of  Physiological  Chemistry,  Yale  University, 
New  Haven,  Connecticut.) 

(Received  for  publication,  October  16,  1912.) 

The  demonstration  of  the  occurrence  of  pyrimidine  derivatives 
as  constituents  of  the  nucleic  acid  molecule  has  awakened  a  wide 
interest  in  the  physiological  behavior  of  the  pyrimidine  ring.  The 
possible  biochemical  significance  of  this  group  is  attested  by  its 
structural  relationship  to  the  purines,  creatinine,  allantoin  and 
other  physiologically  important  compounds.  Although  hydantoin 
and  its  derivatives  have  not  yet  been  found  present  as  constitu- 
ents of  any  tissues  of  the  body,  the  behavior  of  the  hydantoin 
nucleus,  a  structure  similar  to  the  pyrimidine  grouping  but  contain- 
ing one  less  carbon  atom,  deserves  consideration  in  connection 
with  intermediary  metabolism. 


N— C 


C  C 


N— C 
Pyrimidine  nucleus. 


N— C 


N— C 
Hydantoin  nucleus. 


The  close  relationship  between  hydantoin,  allantoin,  creatinine, 
purine  and  imidazole  may  be  seen  by  a  comparison  of  their  struc- 
tural formulae. 


NH— CO    NH— CO  NH5 


NH— CO    NH— CH  N; 


C  =  0 


C  =  0 


C  =  0     C  =  NH  CH 

II                 II              II  II 
NH— CH2  NH— CH— NH    (CH3)N— CH2  N— CH   N  C  N<^ 

Hvdantoin.     Allantoin.         Creatinine.    Imidazole.  Purine. 


CH     C— NH- 

II  II 


347 


348  Behavior  of  Hydantoin  Derivatives 


None  of  these  substances  with  the  possible  exception  of  the 
imidazole  nucleus  have  been  demonstrated  to  be  destroyed  when 
introduced  into  the  organism.  Dakin  and  Wakeman1  have  shown 
by  perfusion  experiments  with  the  liver  that  some  slight  decom- 
position of  histidine,  which  contains  the  imidazole  nucleus,  may 
take  place  with  the  formation  of  acetoacetic  acid,  but  they  con- 
clude that  the  effect  is  too  slight  to  formulate  any  promising  hypoth- 
esis for  the  catabolism  of  histidine.  Feeding  experiments  with 
histidine2  leave  the  fate  of  this  substance  in  the  organism  in 
doubt.  The  failure  of  these  related  compounds  to  experience 
disintegration  in  metabolism  renders  the  behavior  of  hydantoin, 
a  compound  simpler  than  any  of  the  others,  of  particular  interest. 

From  another  viewpoint,  the  behavior  of  hydantoin  seems 
worthy  of  study.    Lusini3  working  with  alloxan  and  alloxantin 

NH— 
I 

reached  the  conclusion  that  the  grouping  C  =  0  functions  to  stim- 
t 

NH— 

ulate  and  then  inhibit  nerve  centers.    It  is,  according  to  Lusini, 

the  ketone-like  group  ^)CO  which  has  the  stimulating  property 

and  an  abundance  of  these  groups  increases  the  toxicity.  More 
recently  Kleiner4  was  unable  to  confirm  Lusini's  conclusions  since 
barbituric  acid,  which  Kleiner  studied  and  which  is  non-toxic, 
contains  the  ketone 'group  and  differs  little  from  the  toxic  sub- 
stance alloxan.  Inasmuch  as  hydantoin  also  contains  the  alleged 
NH— 
I 

toxic  group,  CO     ,  the  question  of  its  toxicity  is  of  interest. 
I 

NH— 

The  present  paper -deals  with  the  behavior  of  hydantoin  and 

1  Dakin  and  Wakeman:  this  Journal,  x,  p.  499,  1912. 

2  Abderhalden  and  Einbeck:  Zeitschr.  f.  physiol.  Chem.,  Ixii,  pp.  322-32, 
1909;  Abderhalden,  Einbeck  and  Schmid:  ibid.,  lxviii,  pp.  395-99, 1910;  Kow- 
alevsky:  Biochem.  Zeitschr.,  xxiii,  pp.  1-4,  1910. 

3  Lusini:  Ann.  di  chim.  e  di  farmacol.,  xxi,  pp.  145-60,  241-57,  xxii,  pp. 
341-51,  385-94,  1895;  Chem.  Centralbl,  i,  p.  1074;  ii,  p.  838,  1895. 

4  Kleiner:  this  Journal,  xi,  pp.  443-70,  1912. 


Howard  B.  Lewis 


349 


the  ethyl  ester  of  hydantoic  acid,  introduced  in  different  ways 
into  the  organism  of  various  species.  The  hydantoic  acid  ester 
was  prepared  from  glycocoll  ester  hydrochloride  and  potassium 
cyanate,  according  to  the  method  of  Harries  and  Weiss.5  On 
evaporating  the  ester  to  dryness  with  concentrated  hydrochloric 
acid,  it  is  converted  to  the  hydantoin.  The  latter  is  then  purified 
by  recrystallization  from  absolute  alcohol. 


NH2  •  CO  •  NH  •  CH2  •  COOC2H5 

Ethyl  ester  of  hydantoic  acid 

Analysis  (Kjeldahl  nitrogen  determination)  of  the  hydantoin  prepared 
gave  the  following  result: 

Calculated  for 
C»H4N202:  Found: 

N   28.00  per  cent.        27.88  per  cent. 

For  the  identification  of  the  hydantoin  in  the  urine,  use  was 
made  of  the  insoluble  benzalhydantoin.  The  urine  was  acidified 
and  evaporated  to  small  volume  on  a  water  bath,  decolorized 
with  animal  charcoal  and  evaporated  to  dryness.  The  product 
was  then  condensed  with  benzaldehyde  in  the  presence  of  glacial 
acetic  acid,  acetic  anhydride  and  dried  sodium  acetate,  as  de- 
scribed by  Wheeler  and  Hoffman.6  For  the  identification  of 
the  ester,  the  urine  was  evaporated  to  dryness  with  concentrated 
hydrochloric  acid  to  convert  the  ester  into  hydantoin,  and  the 
benzal  derivative  was  prepared  as  before. 

The  analytical  procedures  included  the  Kjeldahl-Gunning  meth- 
od for  nitrogen  and  Folin's  methods  for  urea  and  creatinine. 
Blank  experiments  with  hydantoin  showed  that  this  substance  is 
not  attacked  by  Folin's  urea  method. 

In  the  experiments  with  rabbits  the  bladder  was  emptied  by 
pressure  at  the  same  hour  daily.  The  substances,  when  fed,  were 
dissolved  in  water  and  introduced  through  a  gastric  sound.  In 
the  experiments  with  dogs  the  animals  were  catheterized  at  reg- 

5  Harries  and  Weiss:  Ber.  d.  deutsch.  chem.  Gesellsch.,  xxxiii,  p.  3418,  1900. 
8  Wheeler  and  Hoffman:  Amer.  Chem.  Journ.,  xlv,  p.  368,  1911. 


NH— CO 
I 

C  =  0 
I 

NH— CH2 
Hydantoin 


350  Behavior  of  Hydantoin  Derivatives 


ular  twenty-four-hour  intervals.  Here  the  substances  fed  were 
mixed  with  the  food. 

EXPERIMENTS  WITH  HYDANTOIN. 

Rabbit.  I.  Diet,  300  grams  of  carrots  daily.  This  was  com- 
pletely consumed  except  on  the  day  of  the  hydantoin  administra- 
tion when  the  animal  ate  only  270  grams.  No  toxic  effects  of 
any  sort  were  noted.    The  protocol  follows: 


Rabbit;  weight,  1.8  kgms. 


a 

VOLUME 

SPECIFIC  GRAVITY 

TOTAL  N 

« 

p 

UREA  +  NHs-N 

TOTAL  N 

N  NOT 
UREA  +  NHj 

N 

REMARKS 

CC. 

grams 

gram 

per  cent 

gram 

1 

280 

1.012 

0.876 

0.768 

87.6 

0.108 

2 

295 

1.012 

0.918 

0.750 

80.1 

0.168 

3 

245 

1.015 

0.900 

0.756 

84.0 

0.144 

fl.5  gm.  hydantoin 

4 

145 

1.026 

1.308 

0.738 

56.4 

0  570 

\    =  0.42  gram  N,  in  - 

5 

220 

1.016 

0.696 

0.600 

8( 

1.4 

0.096 

[  traperitoneally. 

6 

205 

1.024 

0.690 

0.630 

90.1 

0.060 

About  0.3  gram  of  benzalhydantoin,  after  purification  by  re- 
crystallization  from  alcohol,  was  obtained  from  the  urine  of  the 
fourth  day.  The  benzalhydantoin  isolated  melted  at  217°  and 
when  mixed  with  a  pure  synthetic  sample  did  not  alter  the  melting 
point  of  the  latter. 

No  increase  in  the  urea  +  ammonia  nitrogen  on  the  day  of  the 
injection  was  observed,  although  the  increase  in  the  elimination 
of  total  nitrogen  excreted  accounted  for  all  the  nitrogen  admin- 
istered as  hydantoin.  This  fact,  together  with  the  identification 
of  hydantoin  in  the  urine,  indicates  that  hydantoin  is  unaltered 
in  its  passage  through  the  body. 

Another  experiment  with  the  same  animal  a  few  days  later, 
in  which  the  same  amount  of  hydantoin  was  administered  per  os, 
gave  similar  results. 

Rabbit.    II.    Diet,  300  grams  of  carrots.    On  the  day  on  which 


Howard  13.  Lewis 


35i 


the  hydantoin  was  fed,  the  animal  consumed  the  full  daily  ration. 
No  toxic  symptoms  of  any  sort  were  noted.    The  protocol  follows: 

Rabbit;  weight,  1.64  kgms. 


< 

Q 

VOLUME 

SPECIFIC  GRAVITY 

TOTAL  N 

< 

« 
P 

5 

n 

+ 

H 
K 
P 

>j 

H 
O 
H 

P 

REMARKS 

CC. 

gram 

gram 

per  ceni 

gram 

1 

275 

1.015 

0.900 

0.786 

87.3 

0.114 

2 

240 

1.018 

0.708 

0.642 

90.6 

0.066 

3 

150 

1.016 

0.624 

0.558 

89.4 

0.066 

fl.5  gm.  hydantoin 

4 

205 

1.021 

0.918 

0.576 

1.7 

0  342 

\     =  0.42  gram  N, 

5 

280 

1.019 

0.726 

0.570 

79.1 

0  156 

{    per  os. 

6 

160 

1.020 

0.600 

0.516 

86.0 

0.084 

7 

160 

1.025 

0.570 

0.474 

83.1 

0.096 

From  the  urine  of  the  experimental  day  a  small  amount  of  ben- 
zalhydantoin  melting  at  218°  was  obtained. 

Dog.  A  female  was  fed  on  a  constant  daily  diet  of  200  grams  of 
lean  meat,  50  grams  of  lard,  30  grams  of  sugar,  5  grams  of  bone 
ash,  2  grams  of  salt  and  250  cc.  of  water  with  a  total  nitrogen 
content  of  6.84  grams.  The  hydantoin  was  dissolved  in  the  water 
of  the  diet.  The  animal  ate  eagerly  on  the  experimental  day  as 
at  other  times. 

Dog  A;  weight,  12.4  kgms. 


< 

H 

a 

p 

o 

> 

« 

O 

£ 

o 
w 

H 

M 

B 

K 

« 

pi 

i+* 

« 

H 
g 

g 

-J 

sa 

REMARKS 

o 

> 

Cm 

to 

O 
H 

p 

PS 

o 

cc. 

grams 

grams 

per  cent 

grams 

gram 

1 

160 

1.055 

8.45 

7.63 

89.8 

0.82 

0.279 

2 
3 
4 

180 
300 
300 

1.042 
1.025 
1.030 

6.69 
6.38 
6.87 

6.24 
5.80 
5.67 

93.3 
90.9 
82  6 

0.45 
0.58 
1  20 

0.294 
0.294 
0.298 

2.5    gm.  hy- 
dantoin = 
0.7  gm.  N, 
with  food. 

5 

320 

1.024 

5.99 

5.39 

90.0 

0.60 

0.306 

6 

350 

1.023 

6.12 

5.44 

88.8 

0.68 

0.284 

7 

300 

1.028 

6.37 

5.76 

90.4 

0.61 

0.297 

352  Behavior  of  Hydantoin  Derivatives 


From  the  urine  of  the  experimental  day  3.3  grams  of  benzal- 
hydantoin  were  obtained  corresponding  to  1.7  grams  of  hydantoin 
(  =  0.476  gram  N)  in  the  day's  urine.  This  was  purified  by  solu- 
tion in  potassium  hydroxide  and  reprecipitation  with  acid. 

Cat.  A  cat  weighing  approximately  4  kgms.  received  3  grams 
of  hydantoin  mixed  with  raw  meat.  The  urine  of  the  next  twenty- 
four  hours  was  collected  and  examined  for  the  presence  of  hydan- 
toin as  above.  The  benzalhydantoin  obtained  melted  at  214°  and 
after  purification  weighed  1.7  grams. 

A  nitrogen  determination  (Kjeldahl)  on  the  mixed  products 
obtained  in  this  and  the  three  preceding  experiments  gave  the 
following  results: 

Calculated  for 
C10H8N2O2:  Found: 

N   14.92  per  cent.         14.59  per  cent. 

In  all  our  experiments  the  recovery  of  the  administered  hydan- 
toin from  the  urine  leaves  no  doubt  as  to  its  absorption.  The 
increase  in  the  total  nitrogen  of  the  urine  on  the  experimental 
day  also  points  to  the  same  conclusion.  In  no  case  was  the  urea 
+  ammonia  nitrogen  output  increased,  the  difference  between 
the  total  and  urea  +  ammonia  nitrogen  nearly  always  approach- 
ing the  value  of  the  nitrogen  administered  as  hydantoin.  In 
the  rabbit  II,  there  was  observed  a  slight  lag,  part  of  the  hydantoin 
probably  being  eliminated  on  the  day  after  the  administration. 
No  effect  on  the  creatinine  elimination  was  observed  in  the  dog. 

Hydantoin  appears  to  be  without  influence  on  nitrogenous  me- 
tabolism and  is  not  destroyed  or  changed  by  the  organism.  No 

NH — 
I 

toxicity  attributable  to  the  group  C  =  O  as  alleged  by  I^usini  was 

NH — 

observed. 

EXPERIMENTS  WITH  ETHYL  HYDANTOATE. 

In  order  to  ascertain  whether  the  inability  of  the  organism  to 
break  down  hydantoin  was  due  to  the  cyclic  structure,  experiments 
were  conducted  with  the  ethyl  ester  of  hydantoic  acid.  This 
acid  is  converted  to  hydantoin  with  the  loss  of  a  molecule  of  water 


Howard  13.  Lewis 


353 


and  bears  the  same  relation  to  hydantoin  that  creatine  hears  to 
creatinine. 

NH— CO 


NH2CONH-CH,COOH 


Hydantoic  acid 


c=o 


+  H20 


NH— CH2 
Hydantoin 


Hydantoic  acid  may  also  be  considered  as  a  uramino  acid, 
uraminoacetic  acid.  Koehne,7  working  with  the  ethyl  ester  of  the 
homologous  uraminoformic  or  allophanic  acid,  found  that  it  dis- 
appeared in  the  body. 

Rabbit  1.  Diet,  300  grams  of  carrots  daily.  On  the  exper- 
imental day  the  animal  showed  no  unusual  symptoms  and  resumed 
eating  immediately  after  the  feeding  of  the  ester.  The  protocol 
follows: 

Rabbit;  weight,  1.7  kgms. 


>< 

a 
a 

o 

ECIFIC  GRAVITY 

lEATININE 

ITAL  N 

< 

a 

351 

Aba 
■fJS 

el 

'A  a 

REMARKS 

6 

> 

5 

o 

EH 

8 

cc. 

gram 

gram 

gram 

per  cent 

gram 

1 

220 

1.011 

0.055 

0.810 

0.729 

90.0 

0.091 

2 

220 

1.012 

0.056 

0.443 

0.347 

78.3 

0.096 

r2  gm.  hydan- 

3 

200 

1.013 

0.047 

0.443 

0.396 

89.8 

0.047 

toic  acid  es- 

4 

295 

1.017 

0.071 

0  845 

0.420 

49  6 

0  425 

ter  =  0.38 

5 

220 

1.016 

0.063 

0.458 

0.347 

73.5 

0.111 

gram  N,  per 

6 

270 

1.014 

0.061 

0.414 

0.369 

89.1 

0.045 

OS. 

From  the  urine  of  the  experimental  day,  benzalhydantoin  was 
prepared  in  the  usual  manner.  An  amount  equivalent  to  0.67 
gram  of  the  ester  (=  0.13  gram  N.)  was  obtained.  The  ben- 
zalhydantoin melted  at  218°  and  did  not  change  the  melting  point 
of  the  pure  synthetic  substance  when  mixed  with  it.  A  nitrogen 
(Kjeldahl)  determination  gave  the  following 'results: 

Calculated  for 
C10H8N2O2:  Found: 

N   14.92  per  cent.  14.67  per  cent. 


7  Koehne:  Inaugural  Dissertation,  Rostock,  1894,  p.  17. 


354  Behavior  of  Hydantoin  Derivatives 


Rabbit  2.  Diet,  300  grams  of  carrots  daily.  The  animal  ap- 
peared normal  on  the  day  of  the  injection  and  ate  as  usual. 
The  protocol  follows: 


Rabbit;  weight,  1.5  kgms. 


DAY 

VOLUME 

SPECIFIC 
GRAVITY 

TOTAL  N 

CREATININE 

REMARKS 

CC. 

grams 

gram 

1 

95 

1.026 

1.172 

0.103 

2 

180 

1.020 

0.702 

0.070 

3 

220 

1.013 

0.611 

0.078 

fl.5  grams  hydantoic  acides- 

4 

210 

1.013 

0.878 

0.081 

\    ter  =  0.286  gram  N,  sub- 

5 

240 

1.012 

0.513 

0.084 

[  cutaneously. 

6 

240 

1.012 

0.374 

0.076 

7 

230 

1.016 

0.444 

0.073 

From  the  urine  of  the  experimental  day  a  small  amount  of 
benzalhydantoin  was  prepared  which  melted  at  217°  and  did  not 
affect  the  melting  point  of  the  pure  synthetic  substance. 

Rabbit  3.  Diet,  300  grams  of  carrots  and  30  grams  of  oats 
daily.  On  the  day  of  the  injection  only  270  grams  of  carrots  were 
eaten.    No  abnormal  symptoms  were  noted. 


Rabbit;  weight,  2.34  kgms. 


< 

a 

a 

(3 
O 

ECIFIC  GRAVITY 

< 

H 

< 
« 

A 
+ 

B 
« 

TOTAL  N 

tEATININE 

REMARKS 

Q 

> 

00 

O 
H 

S3 

P 

D 

m 
U 

CC. 

grams 

grams 

per  ce«< 

gram 

gram 

1 

2 

265 
220 

1.010 
1.013 

1.20 

0.855 

1.020 
0.716 

85.0 
82.9 

0.180 

0.139 

0.122 
0.092 

2  gm.  hydan- 
toic acid  es- 
ter =  0.38 
grams  N. 
intr  aperi- 
toneally. 

3 
4 

220 
260 

1.012 
1.015 

1.018 
1.476 

0.840 
1.035 

83.3 
70.3 

0.180 
0.441 

0.110 
0.145 

5 
6 

185 
160 

1.015 
1.015 

0.996 
0.990 

0.852 
0.900 

85.5 
90.9 

0.144 
0.090 

0.123 
0.135 

7 

125 

1.018 

0.945 

0.810 

85.7 

0.135 

0.081 

From  the  urine  of  the  experimental  day  benzalhydantoin  was 
prepared  and  purified  by  solution  in  potassium  hydroxide  and 
reprecipitation  with  acid.    It  weighed  0.75  gram  =  0.58  gram  ester 


Howard  B.  Lewis 


355 


in  the  day's  urine.  A  nitrogen  (Kjeldahl)  determination  gave 
the  following  results. 


N 


Calculated  for 
C10II8N2O2: 

14.92  per  cent. 


Found: 
14.88  per  cent. 


Dog.  A  female  received  a  standard  daily  diet  (see  experiments 
on  hydantoin).  The  food  was  eaten  as  usual  on  the  experimental 
day. 

Dog  A ;  weight,  12.6  kgms. 


< 

0 

s 
p 
J 
0 
> 

SPECIFIC  GRAVITY 

TOTAL  N 

<! 
W 
M 
P 

UREA  +  NHs-N 

TOTAL  N 

N  NOT 
UREA  +  NH3 

N 

CREATININE 

REMARKS 

cc. 

grams 

grams 

per  cent 

grams 

gram 

1 

175 

1.040 

6.61 

6.03 

91 

.2 

0.58 

0.349 

2 

350 

1.026 

6.60 

6.03 

91 

.3 

0.57 

0.335 

3  gm.  hydan- 

3 

380 

1.029 

6.59 

5.93 

90.0 

0.66 

0.324 

toicacid  es- 

4 

300 

1.028 

7  34 

6.25 

85.1 

1.09 

0.326 

ter  =  0.67 

gm.    N  in 

food. 

5 

290 

1.033 

6.25 

5.67 

90.8 

0.58 

0.335 

2  gm.  ester  = 

6 

315 

1.026 

6.53 

5.92 

90.7 

0.61 

0!324 

0.38  gm.  N 

7 

290 

1.034 

6.44 

5.55 

86  1 

0.89 

0.332 

in  food.  See 

note  in  the 

k  text. 

On  the  seventh  day  the  animal  received  a  second  dose  of  2  grams 
of  the  ester,  but  refused  to  eat  all  the  food.  The  animal  was 
stuffed,  and  some  material  was  lost.  Hence  the  urine  data  on 
that  day  cannot  be  compared  with  those  of  the  previous  days. 
Taken  as  an  isolated  experiment,  however,  the  results  of  the  seventh 
day  show  the  important  point  that  the  urea  +  ammonia  nitrogen 
does  not  maintain  its  fairly  uniform  proportion  as  on  other  days 
but  is  decreased,  indicating  excretion  of  nitrogen  in  some  form 
other  than  urea. 

Small  amounts  of  benzalhydantoin  were  prepared  from  the 
urine  of  the  experimental  days.  The  material  was  darker  than 
that  obtained  in  previous  experiments  and  melted  at  210-212°. 
A  nitrogen  (Kjeldahl)  determination  gave  the  following  results: 


N. 


Calculated  for 
C10H8N2O2: 

14.92  per  cent. 


Found: 
14.41  per  cent. 


THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  XIII,  NO.  3. 


356  Behavior  of  Hydantoin  Derivatives 


These  experiments  all  indicate  that  in  the  dog  and  the  rabbit 
the  hydantoic  acid  ester  is  not  affected  by  the  metabolic  processes 
of  the  body.  No  marked  increase  of  urea  and  ammonia  nitrogen 
was  observed.  A  hydantoin  derivative  was  always  isolated  from 
the  urine,  indicating  the  presence  of  the  unaltered  substance 
administered.  The  creatinine  output  of  the  urine  was  not  affected. 
The  very  slight  increase  observed  in  the  experiments,  especially 
with  rabbit  2,  may  be  accounted  for  by  the  fact  that  the  ester 
itself  gives  a  slight  color  with  Jaffe's  picric  acid  test  as  it  is  applied 
in  the  colorimeter. 

SUMMARY. 

« 

1.  After  administration  of  hydantoin,  the  compound  can  be 
recovered  from  the  urine  in  the  form  of  an  insoluble  benzalhy- 
dantoin.  This  method  is  not  quantitative,  but  serves  to  identify 
the  material. 

2.  No  toxic  effects  were  observed  to  follow  the  administration 
of  hydantoin.    This  is  in  opposition  to  Lusini's  theory  of  the 

NH— 
I 

toxicity  of  C  =  0  groups. 
I 

NH— 

3.  Hydantoic  acid,  of  which  hydantoin  is  the  cyclic  anhydride, 
is  not  destroyed  in  metabolism  when  it  is  administered  as  the  ethyl 
ester,  but  can  likewise  be  recovered  by  forming  the  insoluble  ben- 
zalhydantoin. 

4.  The  hydantoin  nucleus  is  not  destroyed  in  the  organism  of 
the  cat,  rabbit  or  dog. 

These  studies  were  undertaken  at  the  suggestion  of  Professor 
Lafayette  B.  Mendel.  We  desire  to  acknowledge  our  indebtedness 
to  Professor  Treat  B.  Johnson  for  aid  in  the  synthesis  of  the  com- 
pounds employed. 


y 


